Peptide sequences for detection and differentiation of antibody responses to sars-cov-2 and other human corona viruses

ABSTRACT

The current disclosure provides compositions, methods, and kits for detecting the exposure to and infection by certain viruses. Specifically, the current disclosure allows for the rapid differential serological detection of exposure to, and infection by viruses. In particular, the current disclosure allows for the rapid differential serological detection of exposure to, and infection by corona virus (SARS-CoV-2). The current disclosure provides for peptides for the differential detection of SARS-CoV-2.

CROSS-REFERENCE TO OTHER APPLICATIONS

The present application claims priority to U.S. patent applications Ser. Nos. 63/005,865 filed Apr. 6, 2020 and 63/064,074 filed Aug. 11, 2020, both of which are hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under AI109761 awarded by the National Institutes of Health. As such, the United States government has certain rights in this invention.

FIELD

This disclosure herein relates to the field of detection of antibody responses and exposure to viruses, in particular the detection of an antibody response and exposure to corona virus (SARS-CoV-2).

BACKGROUND

In December 2019, a cluster of cases were found positive for severe pneumonia-like illness in Wuhan, Hubei Province, China. A cluster of 41 patients that developed severe pneumonia like illness were shown to be infected with a novel coronavirus (nCoV) (Coronaviridae Study Group 2020). Sequence analyses of nCoV indicated it was a beta coronavirus, with high similarity to severe acute respiratory syndrome coronavirus (SARS-CoV). Subsequently, the International Committee on Taxonomy of Viruses (ICTV)/WHO named this virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its disease coronavirus disease 2019 (COVID-19) (Zhou et al. 2020). On Jan. 30, 2020, the WHO declared COVID-19 a global health emergency. Within 12 weeks after identification of first cluster, there were greater than 790,000 positive cases of COVID-19 globally, with 38,000 fatalities (19%). A COVID-19 outbreak began in the United States with an evacuated patient from Wuhan on Jan. 15, 2020. On Feb. 4, 2020, the Secretary of the Department of Health and Human Services (HHS), USA also declared a public health emergency with significant potential to affect national security or the health and security of United States citizens living abroad. As of April 2020, the virus had resulted in over a million cases worldwide, with tens of thousand deaths.

The severity and rapid progression of COVID-19 has impacted the majority of the population through physical, social, and financial distress. The mitigation and containment measures forced global lockdowns, curfews and home isolations, that have already affected global economy to a greater extent than the terrorist attacks of Sep. 11, 2001, or the 2008 financial crisis. It has been estimated the pandemic may cost approximately $3 trillion and result in a loss of 5-10% global GDP

Diagnostic tests are critical for clinical and public health management of COVID-19. These tests fall into two broad categories: molecular assays used to identify infected individuals and to detect environmental contamination; and serological assays used to determine who has been exposed.

Molecular assays for detection of SARS-CoV-2 are useful in diagnosis of active infection but cannot provide insight into historical infection, especially in those individuals with mild or non-existence symptoms.

Additionally, vaccines are at the forefront of COVID-19 research. As these vaccines are developed, there is a need for rapid reliable assays for testing the efficacy of such vaccines.

Thus, there is a need for a sensitive, specific, and inexpensive high throughput serological assay for the diagnosis of SARS-CoV-2 exposure.

SUMMARY

The current disclosure provides compositions, methods, devices, and kits for detecting the exposure to, and infection by, certain viruses. Specifically, the current disclosure allows for the rapid differential serological detection of exposure to, and infection by viruses. In particular, the current disclosure allows for the rapid serological differential detection of exposure to, and infection by corona virus (SARS-CoV-2), and discrimination of antibody responses to linear epitopes specific to seven human coronaviruses: SARS-CoV-2; SARS; MERS; NL-63; OC-43; 229E; and HKU1.

Described herein is the identification of peptides that will enable specific and sensitive serological differential diagnosis of coronavirus infections on a wide range of platforms including but not limited to microarrays, ELISA, RIA, lateral flow, western blot, and bead-based assays. These assays will allow identification of individuals who are immune to future infection, capable of deployment to the workforce and who may serve as donors of antibodies for control of COVID-19. Cross-reactivity between SARS-CoV-2, SARS-CoV, MERS-CoV and seasonal coronaviruses (e.g., OC43, 229E, HKU1, and NL63) complicate differential serodiagnosis and efforts to investigate the epidemiology of infection and linkage to disease. Serology by plaque reduction neutralization test (PRNT) is an option but PRNTs are expensive, labor-intensive and require live virus and BSL3 facility. Furthermore, antibodies to conserved coronaviruses domains may confound assay specificity, particularly in the acute and early convalescent phase.

Described herein is a sensitive, highly multiplexed, microarray-based assay that enabled the discrimination of antibody responses to linear epitopes specific to seven human coronaviruses: SARS-CoV-2: SARS: MERS: NL-63: OC-43: 229E and HKU1. Also disclosed herein is the identification of 29 highly specific IgG epitopes for SARS-CoV-2 (Table 2) as well as additional 163 IgG epitopes for SARS-CoV-2 (Table 4) and 16 highly specific IgM epitopes for SARS-CoV-2 (Table 3). Also disclosed herein are highly specific epitopes for a human coronavirus other than SARS-CoV-2, listed in Table 5.

The compositions, methods, devices, and kits for detection of exposure to, and infection by SARS-CoV-2 comprise specific peptides, isolated and non-isolated, which are strongly reactive with, and specific for SARS-CoV-2, i.e., reactive and specific epitopes of antibodies to SARS-CoV-2. Additionally, the compositions, methods, devices, and kits for differential detection of exposure to, and infection by SARS-CoV-2 comprise specific peptides, isolated and non-isolated, which are strongly reactive with, and specific for SARS-CoV-2, i.e., reactive and specific epitopes of antibodies to SARS-CoV-2, as well as specific peptides, isolated and non-isolated, which are strongly reactive with, and specific for a human coronavirus other than SARS-CoV-2.

Thus, one embodiment of the present disclosure is a peptide, which is reactive with, and specific for SARS-CoV-2 antibodies, listed in Table 2 (SEQ ID NOs: 1-26), Table 3 (SEQ ID NOs: 30-45), and Table 4 (SEQ ID NOs: 46-208). A further embodiment is a peptide, which is reactive with, and specific for a human coronavirus other than SARS-CoV-2, listed in Table 5 (SEQ ID NOs: 209-261).

A further embodiment is a collection or set of peptides comprising at least one peptide which is reactive with, and specific for SARS-CoV-2 antibodies, listed in Table 2 (SEQ ID NOs: 1-26), Table 3 (SEQ ID NOs: 30-45), and Table 4 (SEQ ID NOs: 46-208). A further embodiment is a collection or set of peptides comprising at least one peptide which is reactive with, and specific for a human coronavirus other than SARS-CoV-2, listed in Table 5 (SEQ ID NOs: 209-261).

Yet a further embodiment of the present disclosure are collections or sets of peptides comprising amino acid sequences shifted one residue across one or more peptides chosen from the group consisting of the peptides that are reactive with, and specific for SARS-CoV-2 antibodies listed in listed in Table 2 (SEQ ID NOs: 1-26), Table 3 (SEQ ID NOs: 30-45), and Table 4 (SEQ ID NOs: 46-208). Yet a further embodiment of the present disclosure are collections or sets of peptides comprising amino acid sequences shifted one residue across one or more peptides chosen from the group consisting of the peptides which is reactive with, and specific for a human coronavirus other than SARS-CoV-2, listed in Table 5 (SEQ ID NOs: 209-261).

These various peptides, or collections or sets of peptides can comprise or consist of peptides, that comprise or consist of 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, up to 25 amino acids in length, up to 30 amino acids in length, up to 35 amino acids in length, up to 40 amino acids in length, and up to 50 amino acids in length.

The number of peptides in a used in a collection or set can range from 2 peptides to a number in the thousands to tens of thousands to hundreds of thousands.

In another aspect, the disclosure provides compositions comprising two or more disclosed peptides.

In another aspect, the disclosure provides nucleic acids comprising a sequence encoding a disclosed peptide. In addition, the disclosure provides vectors comprising such nucleic acids, and host cells comprising such vectors. In certain embodiments, the vector is a shuttle vector. In other embodiments, the vector is an expression vector (e.g., a bacterial or eukaryotic expression vector). In certain embodiments, the host cell is a bacterial cell. In other embodiments, the host cell is a eukaryotic cell.

In non-limiting examples, the antibodies to SARS-CoV-2 and/or other human coronaviruses can be detected using any number of immunodetection techniques, which include but are not necessarily limited to microarrays, ELISA, RIA, lateral flow, western blot, bead-based assays, dipstick type of assay or a SNAP test, multiplex antibody detection techniques of various kinds, or any modification of such assays that are suitable for detecting antibodies of interest.

In one embodiment, the immunodetection technique is in the form of a programmable peptide array.

In certain embodiments, peptides are attached to or immobilized on a solid support. In one embodiment, the peptides are attached to a solid support through a metallic nanolayer. In certain embodiments, the solid support is a bead (e.g., a colloidal particle, metallic nanoparticle or nanoshell, or latex bead), a flow path in a lateral flow immunoassay device (e.g., a porous membrane), a blot (e.g., Western blot, a slot blot, or dot blot), a flow path in an analytical or centrifugal rotor, or a tube or well (e.g., in a plate suitable for an ELISA assay or microarray). In certain embodiments, peptides are isolated (e.g., synthetic and/or purified) peptides. In certain embodiments, peptides are conjugated to a ligand. For example, in certain embodiments, the peptides are biotinylated. In other embodiments, the peptides are conjugated to streptavidin, avidin, or neutravidin. In other embodiments, the peptides are conjugated to a carrier protein (e.g., serum albumin, keyhole limpet hemocyanin (KLH), or an immunoglobulin Fc domain).

In another aspect, the disclosure provides devices. In certain embodiments, the devices are useful for performing an immunoassay. For example, in certain embodiments, the device is a lateral flow immunoassay device. In other embodiments, the device is an analytical or centrifugal rotor. In other embodiments, the device is a tube or a well, e.g., in a plate suitable for an ELISA assay or a microarray. In still other embodiments, the device is an electrochemical, optical, or opto-electronic sensor.

In certain embodiments, the device comprises at least one disclosed peptide. In other embodiments, the device comprises a collection or set of the disclosed peptides as described herein. In certain embodiments, the peptides are attached to or immobilized upon the device.

In another aspect, the disclosure provides methods of detecting in a sample an antibody to an epitope of SARS-CoV-2. In certain embodiments, the methods comprise contacting a sample with one or more disclosed peptides and detecting formation of an antibody-peptide complex comprising said peptide, wherein formation of said complex is indicative of the presence of an antibody to an epitope of SARS-CoV-2 antigen in said sample. In certain embodiments, the methods comprise contacting the sample with a collection or set of different peptides described herein. In certain embodiments, the methods comprise contacting the sample with a collection or set of all of the peptides described herein.

In one embodiment, the disclosure provides a method for the serological detection of exposure to and/or infection by SARS-CoV-2, comprising the use of a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof.

In further embodiments, the method for the serological detection of exposure to and/or infection by SARS-CoV-2 in a sample, comprises: contacting the sample with a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof; and detecting the binding between the anti-SARS-CoV-2 antibodies in the sample and the peptide or peptides.

Yet a further embodiment is a method for the differential serological detection of exposure to and/or infection by SARS-CoV-2, comprising the use of a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies and/or other human coronaviruses comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof.

In further embodiments, the method for the differential serological detection of exposure to and/or infection by SARS-CoV-2 in a sample, comprises: contacting the sample with a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies and/or other human coronaviruses comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof; and detecting the binding between the anti-SARS-CoV-2 and/or anti-human coronavirus antibodies in the sample and the peptide or peptides.

In certain embodiments, the peptide or each peptide in the collection or set is an isolated (e.g., synthetic and/or purified) peptide. In certain embodiments, the peptide, peptides or collection or set of peptides is attached to or immobilized upon a solid support. In one embodiment, the peptide, peptides or collection or set of peptides is attached to the solid support through a metallic (e.g., gold) nanolayer. In certain embodiments, the solid support is a bead or plurality of beads (e.g., a colloidal particle, a metallic nanoparticle or nanoshell, or a latex bead), a flow path in a lateral flow immunoassay device (e.g., a porous membrane), a flow path in an analytical or centrifugal rotor, a blot (e.g., Western blot, a slot blot, or dot blot), or a tube or a well (e.g., in a plate suitable for an ELISA assay or microarray). In certain embodiments, the solid support comprises metal, glass, a cellulose-based material (e.g., nitrocellulose), or a polymer (e.g., polystyrene, polyethylene, polypropylene, polyester, nylon, or polysulfone).

A further embodiment is a method for the serological detection of exposure to and/or infection by SARS-CoV-2, comprising the use of a peptide microarray comprising peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof.

In further embodiments, the method for the serological detection of exposure to and/or infection by SARS-CoV-2 in a sample, comprises: contacting the sample with a peptide microarray comprising a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof; and detecting the binding between the anti-SARS-CoV-2 antibodies in the sample and the peptide or peptides in the microarray.

Yet a further embodiment is a method for the differential serological detection of exposure to and/or infection by SARS-CoV-2, comprising the use of a peptide microarray comprising a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies and/or other human coronaviruses comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof.

In further embodiments, the method for the differential serological detection of exposure to and/or infection by SARS-CoV-2 in a sample, comprises: contacting the sample with a peptide microarray comprising a peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies and/or other human coronaviruses comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof; and detecting the binding between the anti-SARS-CoV-2 and/or anti-human coronavirus antibodies in the sample and the peptide or peptides in the microarray.

In certain embodiments, the detecting step comprises performing an ELISA assay. In other embodiments, the detecting step comprises performing a lateral flow immunoassay. In other embodiments, the detecting step comprises performing an agglutination assay. In other embodiments, the detecting step comprises spinning the sample in an analytical or centrifugal rotor. In other embodiments, the detecting step comprises analyzing the sample using a Western blot, a slot blot, or a dot blot. In still other embodiments, the detecting step comprises analyzing the sample with an electrochemical sensor, an optical sensor, or an opto-electronic sensor. In certain embodiments, the detecting step comprises performing a wavelength shift assay.

In some embodiments, the peptides consist of the amino acid sequences SEQ ID NOs: 1-261.

The present disclosure also includes systems and kits for the differential serological detection of exposure to and/or infection by SARS-COV-2.

BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the invention, there are depicted in drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

FIG. 1 shows the multidimensional scaling (MDS) of differential IgM peptide signals in assays of sera from subjects with a history of infection with SARS-CoV-2 (n=100) or without historical exposure to SARS-CoV-2 (controls) (n=32). Based on MDS analysis, samples with exposure of SARS-CoV-2 samples (green triangles) versus controls (red circles) clustered in overlapping groups.

FIG. 2 shows the multidimensional scaling (MDS) of differential IgG peptide signals in assays of sera from subjects with a history of infection with SARS-CoV-2 (n=100) or without historical exposure to SARS-CoV-2 (controls) (n=32). Based on MDS analysis, samples with exposure of SARS-CoV-2 samples (green triangles) versus controls (red circles) clustered into two separate groups.

FIG. 3 is a heatmap showing the proteome-wide linear epitope mapping of SARS-CoV-2-specific IgG antibodies by a HCoV peptide array. X axis represents 981 peptides from SARS-CoV-2 proteins, Y axis represents 132 samples tested using the HCoV peptide array. The heatmap is plotted with normalized values of individual peptide intensity in AU for each of the 132 plasma samples. Panel grids outlined in dotted lines show highly reactive areas in S and N proteins. *ORF1ab protein is large so divided partly in lower panel owing to larger size.

FIG. 4 are graphs of the reactivity intensities of each peptide from 29 immunogenic linear IgG epitopes are plotted on log scale (y-axis). Reactivity to selected peptides is plotted for corresponding groups shown in different colors (x-axis).

DETAILED DESCRIPTION Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

As used herein, the term “sample” means any substance containing or presumed to contain antibodies, in particular those to SARS-CoV-2. The sample can be of natural or synthetic origin and can be obtained by any means known to those of skill in the art. The sample can be a sample of tissue or fluid isolated from a subject including but not limited to, plasma, serum, whole blood, spinal fluid, semen, amniotic fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tissue. Samples can be research, clinical, or environmental. Sample can also be blood products used to transfuse or treat. Samples can also be synthetic and include but are not limited to in vitro cell culture constituents including but not limited to conditioned medium, recombinant cells, and cell components.

As used herein, the term “subject” means any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In the preferred embodiment, the subject is a human being, a pet or livestock animal.

The term “patient” as used in this application means a human subject.

The term “detection”, “detect”, “detecting” and the like as used herein means as used herein means to discover the presence or existence of.

The terms “identification”, “identify”, “identifying” and the like as used herein means to recognize exposure to a specific virus or viruses in sample from a subject.

The term “peptide” includes any sequence of two or more amino acids. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right.

The term “amino acid,” includes the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, omithine, citruline, alpha-methylalanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C₁-C₆) alkyl, phenyl or benzyl ester or amide).

An “antigen” (from antibody-generating) or “immunogen” is a substance that prompts the generation of antibodies and can cause an immune response. They may also be used for diagnostic or patient selection or characterization purposes.

Antibodies (also known as immunoglobulins (Ig)) are globulin proteins that are found in blood or other bodily fluids of vertebrates and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units—each with two large heavy chains and two small light chains—to form, for example, monomers with one unit, dimers with two units or pentamers with five units. Antibodies are produced by B cells. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.

Although the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen. This huge diversity of antibodies allows the immune system to recognize an equally wide diversity of antigens. The part of the antigen recognized by an antibody is termed an “epitope.” These epitopes bind with their antibody in a highly specific interaction, called induced fit, which allows antibodies to identify and bind only their specific epitope in the matching antigen(s) in the midst of the millions of different molecules that make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts of the immune system. Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection. Production of antibodies is the main function of the humoral immune system.

As used herein, the term “isolated” and the like means that the referenced material is free of components found in the natural environment in which the material is normally found. In particular, isolated biological material is free of cellular components. An isolated peptide or protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated material may be, but need not be, purified.

The term “substantially purified,” as used herein, refers to a molecule, such as a peptide, that is substantially free of cellular material (proteins, lipids, carbohydrates, nucleic acids), culture medium, chemical precursors, chemicals used in synthesis of the peptide, or combinations thereof. A peptide that is substantially purified has less than about 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of the cellular material, culture medium, other polypeptides, chemical precursors, and/or chemicals used in synthesis of the peptide. Accordingly, a substantially pure molecule, such as a peptide, can be at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, by dry weight, the molecule of interest.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation.

For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

The current disclosure enhances the differential diagnosis and management of SARS-CoV-2 by establishing a new serologic assay platform for profiling a subject's pathogen exposure history, as well as providing peptides which are reactive with and sensitive to antibodies to SARS-CoV-2.

In contrast to molecular diagnostics where advances in technology such as polymerase chain reaction and high-throughput screening have dramatically improved sensitivity, specificity and breadth over the past 20 years, serologic methods remained largely unchanged. This lag is important given the role of serology in establishing the distribution and frequency of infection, testing the significance of association between the finding of an agent and disease, and in focusing efforts in pathogen discovery. Described herein is a sensitive, unbiased, highly multiplexed platform for diagnostic serology of SARS-CoV-2 in particular. This peptide array-based platform will enable new strategies for investigating the epidemiology and pathogenesis of acute and chronic diseases due to infection and for monitoring humoral responses to vaccines and immunomodulatory drugs. It will also serve as a screening tool for rapid selection of key informative peptides that can be used in established, inexpensive, alternative platforms including lateral flow immunoassays. Such applications will have practical utility for both clinical medicine and public health by enabling retrospective differential diagnosis of an infectious illness (when genetic footprints of the agent may no longer be present), and in facilitating outbreak investigation and surveillance.

This is particularly useful in infection by SARS-CoV-2 as a patient could no longer have the evidence of acute infection, e.g., viral nucleic acids, but have had a SARS-CoV-2 infection in the past. These assays will allow identification of individuals who are immune to future infection, capable of deployment to the workforce and who may serve as donors of antibodies for control of COVID-19, as well as for testing the effectiveness of vaccines for SARS-CoV-2.

Additionally, serodiagnosis of SARS-CoV-2 infection can be impeded by immunological cross-reactivity among the human coronaviruses (HCoVs): SARS-CoV-2;

SARS-CoV-1; MERS-CoV; OC43; 229E; HKU1; and NL63. Disclosed herein are SARS-CoV-2 peptides that enable discrimination between exposure to SARS-CoV-2 and other HCoVs.

A high-density peptide microarray and plasma samples collected at two time points from 50 subjects with SARS-CoV-2 infection confirmed by qPCR, samples collected in 2004-2005 from 11 subjects with IgG antibodies to SARS-CoV-1, 11 subjects with IgG antibodies to other seasonal human coronaviruses (HCoV), and 10 healthy human subjects were used. Through statistical modeling with linear regression and multidimensional scaling specific peptides were identified that were reassembled to identify 29 linear IgG SARS-CoV-2 epitopes that were immunoreactive with plasma from individuals who had asymptomatic, mild or severe SARS-CoV-2 infections, as well as 16 IgM SARS-CoV-2 epitopes and an additional 163 IgG epitopes for SARS-CoV-2. Also disclosed herein are highly specific epitopes for a human coronavirus other than SARS-CoV-2.

Thus disclosed herein is a sensitive, highly multiplexed, microarray-based assay that enabled the discrimination of antibody responses to linear epitopes specific to seven human coronaviruses: SARS-CoV-2; SARS; MERS; NL-63; OC-43; 229E; and HKU1 and the identification of 29 highly specific IgG epitopes, 16 IgM epitopes for SARS-CoV-2, and additional 163 IgG epitopes for SARS-CoV-2 and 53 highly specific epitopes for a human coronavirus other than SARS-CoV-2.

The work reported herein has been published in recently (see Mishra et, Immunoreactive peptide maps of SARS-CoV-2, Commun Biol 4, 225 (2021) (herein incorporated by reference in its entirety)).

Isolated Peptides Specific and Sensitive for SARS-COV-2 Antibodies (SARS-COV-2 Reactive Peptides) and Other Human Coronavirus Antibodies (Human Coronavirus Reactive Peptides)

The present disclosure includes isolated peptides which are strongly reactive with, and sensitive to antibodies to SARS-CoV-2 in a patient sample. These peptides can be used in any type of serological assay or platform, now known or later developed, to screen for the presence of antibodies to SARS-CoV-2 and to determine if a subject has had an infection by and/or exposure to SARS-COV-2. These peptides can also be used to test for and monitor humoral responses to vaccines and immunomodulatory drugs, thus, being useful for the development of treatment and preventative agents for SARS-CoV-2.

One embodiment is an isolated peptide highly specific for SARS-CoV-2 listed in Table 2 (SEQ ID NOs: 1-29). A further embodiment is an isolated peptide highly specific for SARS-CoV-2 listed in Table 3 (SEQ ID NOs: 30-46). A further embodiment is an isolated peptide highly specific for SARS-CoV-2 listed in Table 4 (SEQ ID NOs: 46-208).

In further embodiments, isolated peptides highly specific for other human coronaviruses are also provided for herein. Isolated peptides highly specific for human coronavirus HKU1 are listed in Table 5 (SEQ ID NOs: 209-223). Isolated peptides highly specific for human coronavirus NL63 are listed in Table 5 (SEQ ID NOs: 224-233). Isolated peptides highly specific for human coronavirus OC43 are listed in Table 5 (SEQ ID NOs: 234-247). Isolated peptides highly specific for human coronavirus 229E are listed in Table 5 (SEQ ID NOs: 248-252). Isolated peptides highly specific for human coronavirus SARS-CoV-1 are listed in Table 5 (SEQ ID NOs: 253-261).

A further embodiment comprises a collection or set of peptides comprising amino acid sequences shifted one residue across any of the peptides listed in Table 2 (SEQ ID NOs: 1-26), Table 3 (SEQ ID NOs: 30-45), Table 4 (SEQ ID NOs: 46-208), and Table 5 (SEQ ID NOs: 209-261). This collection or set can contain peptides that are 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, and up to 12 amino acids in length.

In one embodiment, each peptide in the set or collection is 12 amino acids in length. Other embodiments include a collection or set of isolated peptides comprising or consisting of amino acid sequences shifted one residue across one, two, three, four, five, six, seven, eight, nine, ten, eleven or all twelve of these twelve peptides. This collection or set can comprise or consist of isolated peptides comprising or consisting of 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, and up to 12 amino acids in length.

Peptides of 12 amino acids were constructed based upon work that shows that antibodies bind to linear peptide sequences ranging from 5 to 9 amino acids in length and bind most efficiently when targets are flanked by additional amino acids (Buus et al. 2012). However, peptides containing less than 12 amino acids in length and more than 12 amino acids in length can be used. Peptides 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, up to 25 amino acids in length, up to 30 amino acids in length, up to 35 amino acids in length, up to 40 amino acids in length, and up to 50 amino acids in length can be used.

In certain embodiments, the peptides described herein are produced by synthetic chemistry (i.e., a “synthetic peptide”). In other embodiments, peptides of the invention are produced biologically. An isolated peptide of the invention can be in water, a buffer, or in a dry form awaiting reconstitution, e.g., as part of a kit. An isolated peptide of the present invention can be in the form of a pharmaceutically acceptable salt. Suitable acids and bases that are capable of forming salts with the peptides of the present invention are well known to those of skill in the art and include inorganic and organic acids and bases.

In certain embodiments, the peptides described herein are modified. The peptides may be modified by a variety of techniques, such as by denaturation with heat and/or a detergent (e.g., SDS). Alternatively, peptides may be modified by association with one or more further moieties. The association can be covalent or non-covalent, and can be, for example, via a terminal amino acid linker, such as lysine or cysteine, a chemical coupling agent, or a peptide bond. The additional moiety can be, for example, a ligand, a ligand receptor, a fusion partner, a detectable label, an enzyme, or a substrate that immobilizes the peptide.

The disclosed peptides can be conjugated to a ligand, such as biotin (e.g., via a cysteine or lysine residue), a lipid molecule (e.g., via a cysteine residue), or a carrier protein (e.g., serum albumin, immunoglobulin Fc domain, keyhole limpet hemocyanin (KLH) via e.g., a cysteine or lysine residue). Attachment to ligands, such as biotin, can be useful for associating the peptide with ligand receptors, such as avidin, streptavidin, polymeric streptavidin, or neutravidin. Avidin, streptavidin, polymeric streptavidin, or neutravidin, in turn, can be linked to a signaling moiety (e.g., an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (ALP), or other moiety that can be visualized, such as a metallic nanoparticle or nanoshell (e.g., colloidal gold) or a fluorescent moiety), or a solid substrate (e.g., nitrocellulose membrane). Alternatively, the peptides of the invention can be fused or linked to a ligand receptor, such as avidin, streptavidin, polymeric streptavidin, or neutravidin, thereby facilitating the association of the peptides with the corresponding ligand, such as biotin and any moiety (e.g., signaling moiety) or solid substrate attached thereto. Examples of other ligand-receptor pairs are well-known in the art and can similarly be used.

The peptides can be fused to a fusion partner (e.g., a peptide or other moiety) that can be used to improve purification, to enhance expression of the peptide in a host cell, to aid in detection, and to stabilize the peptide. Examples of suitable compounds for fusion partners include carrier proteins (e.g., serum albumin, immunoglobulin Fc domain, KLH), and enzymes (e.g., horse radish peroxidase (HRP), beta-galactosidase, glutathione-S-transferase, alkaline phosphatase). The fusion can be achieved by means of a peptide bond. For example, peptides of the invention and fusion partners can be fusion proteins and can be directly fused in-frame or can comprise a peptide linker.

In addition, the disclosed peptides may be modified to include any of a variety of known chemical groups or molecules. Such modifications include, but are not limited to, glycosylation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment to polyethylene glycol (e.g., PEGylation), covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, modifications with fatty acids, and transfer-RNA mediated addition of amino acids to proteins such as arginylation. Analogues of an amino acid (including unnatural amino acids) and peptides with substituted linkages are also included.

The disclosed peptides that consist of any of the sequences discussed herein may be modified by any of the discussed modifications. Such peptides still “comprise” or “consist of” the amino acids.

Modifications as set forth above are well-known to those of skill in the art and have been described in great detail in the scientific literature.

Nucleic Acids Comprising a Sequence Encoding the SARS-CoV-2 Reactive Peptides and Other Human Coronavirus Reactive Peptides

In another aspect, the disclosure provides nucleic acids comprising a sequence encoding any disclosed peptide. Nucleic acids of the invention can be single- or double-stranded. A nucleic acid can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof. The nucleic acids can be purified free of other components, such as proteins, lipids and other polynucleotides. For example, the nucleic acids can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. The nucleic acids encode the peptides described herein. In certain embodiments, the nucleic acids encode a peptide having the sequence listed in Table 2 (SEQ ID NOs: 1-26), Table 3 (SEQ ID NOs: 30-45), Table 4 (SEQ ID NOs: 46-208) and/or Table 5 (SEQ ID NOs: 209-261). Nucleic acids can comprise other nucleotide sequences, such as sequences coding for linkers, signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands useful in protein purification such as glutathione-S-transferase, histidine tag, and staphylococcal protein A.

Methods for preparing polynucleotides operably linked to an expression control sequence and expressing them in a host cell are well-known in the art. See, e.g., U.S. Pat. No. 4,366,246. A nucleic acid of the invention is operably linked when it is positioned adjacent to or close to one or more expression control elements, which direct transcription and/or translation of the polynucleotide.

Thus, for example, the peptides described herein can be produced recombinantly following conventional genetic engineering techniques. To produce a recombinant peptide, a nucleic acid encoding the peptide is inserted into a suitable expression system. Generally, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected peptide is operably linked to an expression control sequence permitting expression of the peptide. Numerous types of appropriate expression vectors are known in the art, including, e.g., vectors containing bacterial, viral, yeast, fungal, insect or mammalian expression systems. Methods for obtaining and using such expression vectors are well-known. For guidance in this and other molecular biology techniques used for compositions or methods of the invention, see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, New York; Miller et al, Genetic Engineering, 8:277-298 (Plenum Press, current edition), Wu et al., Methods in Gene Biotechnology (CRC Press, New York, N.Y., current edition), Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., current edition), and Current Protocols in Molecular Biology, (Ausabel et al., Eds.,) John Wiley & Sons, NY (current edition), and references cited therein.

Accordingly, the disclosure also provides vectors comprising nucleic acids described herein, and host cells comprising such vectors. In certain embodiments, the vector is a shuttle vector. In other embodiments, the vector is an expression vector (e.g., a bacterial or eukaryotic expression vector). In certain embodiments, the host cell is a bacterial cell. In other embodiments, the host cell is a eukaryotic cell.

Platforms, Assays and Device for Using the SARS-CoV-2 Reactive Peptides and Other Human Coronavirus Reactive Peptides

There are a number of different conventional assays for detecting formation of an antibody-peptide complex comprising a peptide or peptides of the invention. For example, the detecting step can comprise performing a microarray assay, an ELISA assay, performing an immunofluorescence assay, performing a lateral flow immunoassay, performing an agglutination assay, performing a wavelength shift assay, performing a Western blot, slot blot, or dot blot, analyzing the sample in an analytical or centrifugal rotor, or analyzing the sample with an electrochemical, optical, or opto-electronic sensor. These different assays are described herein and/or are well-known to those skilled in the art.

Thus, the peptides disclosed herein can be used in any assay, format or platform for antibody detection including but not limited to microarrays, ELISA, RIA, lateral flow, western blot, and bead-based assays, as well as those platforms that are later developed.

In certain embodiments, the assay comprises immobilizing the antibody(s) in the sample; adding a peptide disclosed herein; and detecting the degree of antibody bound to the peptide, e.g., by the peptide being labeled or by adding a labeled substance, such as a labeled binding partner (e.g., streptavidin-HRP or streptavidin-colloidal gold complex) or a labeled antibody which specifically recognizes the peptide.

In other embodiments, the assay comprises immobilizing a peptide disclosed herein; adding the sample containing antibodies; and detecting the amount of antibody bound to the peptide, e.g., by adding another peptide disclosed herein conjugated, directly or indirectly, to a label (e.g., metallic nanoparticle or metallic nanoshell, fluorescent label, or enzyme (e.g., horseradish peroxidase or alkaline phosphatase)) or by adding a labeled substance, such as a binding partner or a labeled antibody which specifically recognizes the sample antibodies (e.g., anti-human IgG antibodies, or anti-human IgM antibodies).

In other embodiments, the assay comprises immobilizing a peptide disclosed herein; adding the sample containing antibodies; and detecting the amount of antibody bound to the peptide, e.g., by adding a first binding partner which specifically recognizes the sample antibodies (e.g., anti-human IgG antibodies, or anti-human IgM antibodies), and further adding a second binding partner, wherein the second binding partner is labeled and recognizes said first binding partner.

In still other embodiments, the assay comprises: reacting the peptide and the sample containing antibodies without any of the reactants being immobilized, and then detecting the amount of complexes of antibody and peptide, e.g., by the peptide being labeled or by adding a labeled substance, such as a labeled binding partner (e.g., streptavidin-HRP or streptavidin-colloidal gold complex) or a labeled antibody which specifically recognizes the peptide.

Immobilization of a peptide can be either covalent or non-covalent, and the non-covalent immobilization can be non-specific (e.g., non-specific binding to a polystyrene surface in a microtiter well). Specific or semi-specific binding to a solid or semi-solid carrier, support or surface, can be achieved by the peptide having, associated with it, a moiety which enables its covalent or non-covalent binding to the solid or semi-solid carrier, support or surface. For example, the moiety can have affinity to a component attached to the carrier, support or surface. In this case, the moiety may be, for example, a biotin or biotinyl group or an analogue thereof bound to an amino acid group of the peptide, and the component is then avidin, streptavidin, neutravidin, or an analogue thereof.

Suitable carriers, supports, and surfaces include, but are not limited to, metallic nanolayers, beads (e.g., magnetic beads, colloidal particles or metallic nanoparticles or nanoshells, such as colloidal gold, or particles or nanoparticles comprising silica, latex, polystyrene, polycarbonate, or PDVF), latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass, gelatin or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or Fab fragments of such antibodies.

The protocols for immunoassays using antigens for detection of specific antibodies are well known in art. For example, a conventional sandwich assay can be used, or a conventional competitive assay format can be used.

Devices for performing specific binding assays, especially immunoassays, are known and can be readily adapted for use in the present methods. Solid-phase assay devices include microtiter plates, flow-through assay devices (e.g., lateral flow immunoassay devices), dipsticks, and immunocapillary or immunochromatographic immunoassay devices.

In embodiments, the solid or semi-solid surface or carrier is the floor or wall in a microtiter well, a filter surface or membrane (e.g., a nitrocellulose membrane or a PVDF (polyvinylidene fluoride) membrane), a hollow fiber, a beaded chromatographic medium (e.g., an agarose or polyacrylamide gel), a magnetic bead, a fibrous cellulose matrix, an HPLC matrix, an FPLC matrix, a substance having molecules of such a size that the molecules with the peptide bound thereto, when dissolved or dispersed in a liquid phase, can be retained by means of a filter, a substance capable of forming micelles or participating in the formation of micelles allowing a liquid phase to be changed or exchanged without entraining the micelles, a water-soluble polymer, or any other suitable carrier, support or surface.

In some embodiments, the peptide is provided with a suitable label which enables detection. Conventional labels may be used which are capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Suitable labels include, but are not limited to, enzymes (e.g., HRP, beta-galactosidase, or alkaline phosphatase), fluorescent labels, radioactive labels, colored latex particles, and metal-conjugated labels (e.g., metallic nanolayers, metallic nanoparticle- or metallic nanoshell-conjugated labels). Suitable metallic nanoparticle or metallic nanoshell labels include, but are not limited to, gold particles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells. Metallic nanolayers suitable for detectable layers include nanolayers comprised of cadmium, zinc, mercury, and noble metals, such as gold, silver, copper, and platinum.

Suitable detection methods include, but are not limited to, detection of an agent which is tagged, directly or indirectly, with a colorimetric assay (e.g., for detection of HRP or beta-galactosidase activity), visual inspection using light microscopy, immunofluorescence microscopy, including confocal microscopy, or by flow cytometry (FACS), autoradiography (e.g., for detection of a radioactively labeled agent), electron microscopy, immunostaining, subcellular fractionation, or the like. In one embodiment, a radioactive element (e.g., a radioactive amino acid) is incorporated directly into a peptide chain. In another embodiment, a fluorescent label is associated with a peptide via biotin/avidin interaction, association with a fluorescein conjugated antibody, or the like. In one embodiment, a detectable specific binding partner for the antibody is added to the mixture. For example, the binding partner can be a detectable secondary antibody or other binding agent (e.g., protein A, protein G, protein L or combinations thereof) which binds to the first antibody. This secondary antibody or other binding agent can be labeled with, for example, a radioactive, enzymatic, fluorescent, luminescent, metallic nanoparticle or metallic nanoshell (e.g. colloidal gold), or other detectable label, such as an avidin/biotin system. In another embodiment, the binding partner is a peptide disclosed herein, which can be conjugated directly or indirectly to an enzyme, such as horseradish peroxidase or alkaline phosphatase or other signaling moiety. In such embodiments, the detectable signal is produced by adding a substrate of the enzyme that produces a detectable signal, such as a chromogenic, fluorogenic, or chemiluminescent substrate.

In some embodiments, the detection procedure comprises visibly inspecting the antibody-peptide complex for a color change or inspecting the antibody-peptide complex for a physical-chemical change. Physical-chemical changes may occur with oxidation reactions or other chemical reactions. They may be detected by eye, using a spectrophotometer, or the like.

One assay format is a lateral flow immunoassay format. Antibodies to human or animal immunoglobulins, can be labeled with a signal generator or reporter (e.g., colloidal gold) that is dried and placed on a glass fiber pad (sample application pad or conjugate pad). The diagnostic peptide is immobilized on membrane, such as nitrocellulose or a PVDF (polyvinylidene fluoride) membrane. When a sample is applied to the sample application pad (or flows through the conjugate pad), it dissolves the labeled reporter, which then binds to all antibodies in the sample. The resulting complexes are then transported into the next membrane (PVDF or nitrocellulose containing the diagnostic peptide) by capillary action. If antibodies against the diagnostic peptide are present, they bind to the diagnostic peptide striped on the membrane, thereby generating a signal (e.g., a band that can be seen or visualized). An additional antibody specific to the labeled antibody or a second labeled antibody can be used to produce a control signal.

An alternative format for the lateral flow immunoassay comprises the peptides or compositions being conjugated to a ligand (e.g., biotin) and complexed with labeled ligand receptor (e.g., streptavidin-colloidal gold). The labeled peptide complexes can be placed on the sample application pad or conjugate pad. Anti-human IgG/IgM or anti-animal IgG/IgM antibodies are immobilized on a membrane, such as nitrocellulose of PVDF, at a test site. When sample is added to the sample application pad, antibodies in the sample react with the labeled peptide complexes such that antibodies that bind to peptides become indirectly labeled. The antibodies in the sample are then transported into the next membrane (PVDF or nitrocellulose containing the diagnostic peptide) by capillary action and bind to the immobilized anti-human IgG/IgM or anti-animal IgG/IgM antibodies. If any of the sample antibodies are bound to the labeled peptides, the label associated with the peptides can be seen or visualized at the test site.

Another assay for the screening of blood products or other physiological or biological fluids is an enzyme linked immunosorbent assay, i.e., an ELISA. Typically in an ELISA, isolated peptides or collection or set of peptides disclosed herein, are adsorbed to the surface of a microtiter well directly or through a capture matrix (e.g., an antibody). Residual, non-specific protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent). The well is then incubated with a biological sample suspected of containing specific antibody. The sample can be applied neat, or more often it can be diluted, usually in a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. After incubating for a sufficient length of time to allow specific binding to occur, the well is washed to remove unbound protein and then incubated with an optimal concentration of an appropriate anti-immunoglobulin antibody that is conjugated to an enzyme or other label by standard procedures and is dissolved in blocking buffer. The label can be chosen from a variety of enzymes, including horseradish peroxidase (HRP), beta-galactosidase, alkaline phosphatase (ALP), and glucose oxidase. Sufficient time is allowed for specific binding to occur again, then the well is washed again to remove unbound conjugate, and a suitable substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally (measured at an appropriate wave length). Conditions for performing ELISA assays are well-known in the art.

In another embodiment of an ELISA, a peptide or a collection or set of peptides disclosed herein is immobilized on a surface, such as a ninety-six-well ELISA plate A sample is then added and the assay proceeds as above.

In still other embodiments, a peptide or collection or set of peptides disclosed herein are electro- or dot-blotted onto nitrocellulose paper. Subsequently, a sample, such as a biological fluid (e.g., serum or plasma) is incubated with the blotted antigen, and antibody in the biological fluid is allowed to bind to the antigen(s). The bound antibody can then be detected, e.g., by standard immunoenzymatic methods or by visualization using metallic nanoparticles or nanoshells coupled to secondary antibodies or other antibody binding agents or combinations thereof.

It should be understood by one of skill in the art that any number of conventional protein assay formats, particularly immunoassay formats, may be designed to utilize the isolated peptides, and collections and sets of peptides disclosed herein, for the detection of SARS-CoV-2 and/or other human coronavirus antibodies in a subject. Thus, the use of the peptides is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats that are known to those of skill in the art. To date most serology has been performed using singleplex ELISA, complement fixation or neutralization assays. More recently, Luminex-based systems have been employed that can address up to 100 antigenic targets simultaneously (i.e., 100 individual pathogens, 100 individual antigenic targets for one pathogen, or some variation thereof). Additionally, arrays are established that comprise spotted recombinant proteins expressed in vitro in E. coli, S. cerevesiae, baculoviruses, or cell-free, coupled transcription-translation.

One goal of the present disclosure is to automate the process of SARS-CoV-2 antibody detection and make it inexpensive, quick and accurate as well as detect exposure per se rather than to rigorously characterize humoral responses to specific pathogens.

One assay that meets these requirements is a programmable peptide array.

The peptide array capacity can be exploited to print multiple arrays per glass slide (configurations of 1, 3, 8, or 12 arrays can be printed).

Thus, one embodiment is a peptide microarray comprising peptides that are reactive with, and specific for SARS-CoV-2 antibodies. In some embodiments the peptide microarray comprises: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof.

A further embodiment is a peptide microarray comprising peptide or peptides which are reactive with, and specific for SARS-CoV-2 antibodies and/or other human coronaviruses comprising: a peptide or peptides chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof.

In some embodiments the peptide array comprises a set or collection of peptides, comprising amino acid sequences shifted one residue across all of the peptides comprising or consisting of the amino acid sequences of SEQ ID NOs: 1-29 and/or SEQ ID NOs: 30-45 and/or SEQ ID NOs: 46-208 and/or SEQ ID NOs: 209-261.

Methods and Systems for Serological Detection of Exposure to SARS-CoV-2

The present disclosure also includes methods and systems for the detection of exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2, in any sample utilizing the various peptides, isolated and non-isolated, and peptide microarrays disclosed herein.

Suitable methods typically include: receiving or obtaining (e.g., from a patient) a sample of bodily fluid or tissue likely to contain antibodies; contacting (e.g., incubating or reacting) a sample to be assayed with a peptide or peptides of the invention, under conditions effective for the formation of a specific peptide-antibody complex (e.g., for specific binding of the peptide to the antibody); and assaying the contacted (reacted) sample for the presence of an antibody-peptide reaction (e.g., determining the amount of an antibody-peptide complex). The presence of an elevated amount of the antibody-peptide complex indicates that the subject was exposed to and infected by SARS-CoV-2. A peptide, including a modified form thereof, which “binds specifically” to an antibody against a SARS-CoV-2 antigen interacts with the antibody, or forms or undergoes a physical association with it, in an amount and for a sufficient time to allow detection of the antibody.

Conditions for reacting peptides and antibodies so that they react specifically are well-known to those of skill in the art. See, e.g., Current Protocols in Immunology (Coligan et al., editors, John Wiley & Sons, Inc.).

Once the disclosed peptide or peptides and sample antibody are permitted to react in a suitable medium, an assay is performed to determine the presence or absence of an antibody-peptide reaction. Any of the assays discussed herein can be used.

The methods and systems of the present disclosure may be used to detect exposure to antigens of SARS-CoV-2 in research and clinical settings.

One sample for use in the methods is a biological sample. A biological sample may be obtained from a tissue of a subject or bodily fluid from a subject including but not limited to nasopharyngeal aspirate, blood, cerebrospinal fluid, saliva, serum, plasma, urine, sputum, bronchial lavage, pericardial fluid, or peritoneal fluid, or a solid such as feces. The subject may be any animal, particularly a vertebrate and more particularly a mammal, including, without limitation, a cow, dog, human, monkey, mouse, pig, or rat. In one embodiment, the subject is a human.

A sample may also be a research, clinical, or environmental sample, such as cells, cell culture, cell culture medium, and compositions for use as, or the development of pharmaceutical and therapeutic agents.

Additional applications include, without limitation, detection of the screening of blood products (e.g., screening blood products for infectious agents), biodefense, food safety, environmental contamination, forensics, and genetic-comparability studies. The present disclosure also provides methods and systems for detecting viral antibodies in cells, cell culture, cell culture medium and other compositions used for the development of pharmaceutical and therapeutic and immunomodulatory agents.

The subject may have been exposed to antigens of SARS-CoV-2, suspected of having exposure to antigens of SARS-CoV-2 or believed not to have had exposure to antigens of SARS-CoV-2.

In one embodiment, the subject is being tested for use of their plasma for treatment of COVID-19 patients.

In one embodiment, the subject may be a test subject, which has been administered a SARS-CoV-2 vaccine or immunomodulatory agent.

The systems and methods described herein support the detection and measure of a humoral immune response to SARS-CoV-2.

One embodiment provides a system for the detection of exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2, in any sample. The system includes at least one subsystem, wherein the subsystem includes a peptide or peptides or a collection or set of peptides disclosed herein, which are reactive with, and specific for SARS-CoV-2 antibodies, comprising: a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof. The system can also include additional subsystems for the purpose of: preparation of the sample; binding of any SARS-CoV-2 antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.

A further embodiment provides a system for the detection of exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2, in any sample. The system includes at least one subsystem, wherein the subsystem includes a peptide microarray comprising a peptide or peptides or a collection or set of peptides disclosed herein, which are reactive with, and specific for SARS-CoV-2 antibodies comprising: a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof. The system can also include additional subsystems for the purpose of: preparation of the sample; binding of any SARS-CoV-2 antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.

Another embodiment provides a system for the differential detection of exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2 and other human coronaviruses, in any sample. The system includes at least one subsystem, wherein the subsystem includes a peptide or peptides or a collection or set of peptides disclosed herein, which are reactive with, and specific for SARS-CoV-2 and other human coronavirus antibodies, comprising: a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof. The system can also include additional subsystems for the purpose of: preparation of the sample; binding of any antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.

A further embodiment provides a system for the differential detection of exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2 and other human coronaviruses, in any sample. The system includes at least one subsystem, wherein the subsystem includes a peptide microarray comprising a peptide or peptides or a collection or set of peptides disclosed herein, which are reactive with, and specific for SARS-CoV-2 and other human coronavirus antibodies comprising: peptide or peptides or a collection or set of peptides disclosed herein, which are reactive with, and specific for SARS-CoV-2 and other human coronavirus antibodies, comprising: a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof. The system can also include additional subsystems for the purpose of: preparation of the sample; binding of any antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.

The present disclosure provides a method for detecting the exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2, in any sample, including the steps of: contacting the sample with a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof, under conditions sufficient for any antibodies to SARS-CoV-2 in the sample and the peptides to bind; and visualizing and/or quantifying any bound antibody or antibodies to the peptides. The method may optionally include a step for washing any unbound sample.

The present disclosure provides a method for differentially detecting the exposure to antigens of SARS-CoV-2 and other human coronaviruses, i.e., antibodies to SARS-CoV-2 and other human coronaviruses, in any sample, including the steps of: contacting the sample with a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof, under conditions sufficient for any antibodies to SARS-CoV-2 and/or other human coronaviruses in the sample and the peptides to bind; and visualizing and/or quantifying any bound antibody or antibodies to the peptides. The method may optionally include a step for washing any unbound sample.

The present disclosure further provides a method for detecting the exposure to antigens of SARS-CoV-2, i.e., antibodies to SARS-CoV-2, in any sample, including the steps of: contacting the sample with a peptide microarray comprising a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); or combinations thereof, under conditions sufficient for any antibodies to SARS-CoV-2 in the sample and the peptides to bind; and visualizing and/or quantifying any bound antibody or antibodies to the peptides. The method may optionally include a step for washing any unbound sample.

The present disclosure further provides a method for differentially detecting the exposure to antigens of SARS-CoV-2 and other human coronaviruses, i.e., antibodies to SARS-CoV-2 and other human coronaviruses, in any sample, including the steps of: contacting the sample with a peptide microarray comprising a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof, under conditions sufficient for any antibodies to SARS-CoV-2 and/or other human coronaviruses in the sample and the peptides to bind; and visualizing and/or quantifying any bound antibody or antibodies to the peptides. The method may optionally include a step for washing any unbound sample.

Any method of detection discussed herein or known in the art can be used for visualizing and/or quantifying the bound antibodies.

Kits

The present disclosure also includes reagents and kits for practicing the methods of the invention. These reagents and kits may vary.

One reagent of the kit would be a peptide or peptides or a collection or set of peptides disclosed herein, which are reactive with, and specific for SARS-CoV-2 and/or other human coronavirus antibodies, comprising: a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof.

In certain embodiments, the peptides are attached to or immobilized on a solid support. In some embodiments, the peptides are attached to or immobilized on a solid support through a metallic nanolayer (e.g., cadmium, zinc, mercury, gold, silver, copper, or platinum nanolayer). In certain embodiments, the solid support is a bead (e.g., a colloidal particle or a metallic nanoparticle or nanoshell), a flow path in a lateral flow immunoassay device, a flow path in an analytical or centrifugal rotor, a tube or a well (e.g., in a plate), or a sensor (e.g., an electrochemical, optical, or opto-electronic sensor).

Reagents for particular types of assays can also be provided in kits disclosed herein. Thus, the kits can include a population of beads (e.g., suitable for an agglutination assay or a lateral flow assay), or a plate (e.g., a plate suitable for an ELISA assay). In other embodiments, the kits comprise a device, such as a lateral flow immunoassay device, an analytical or centrifugal rotor, a Western blot, a dot blot, a slot blot, or an electrochemical, optical, or opto-electronic sensor.

In one particular embodiment, the kit would comprise peptide microarrays comprising a peptide or peptides or a collection or set of peptides, which are reactive with, and specific for SARS-CoV-2 and/or other human coronavirus antibodies, comprising: a peptide or peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a peptide or peptides chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a peptide or peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 2 (SEQ ID NOs: 1-29); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 3 (SEQ ID NOs: 30-45); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 4 (SEQ ID NOs: 46-208); and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of the peptides listed in Table 5 (SEQ ID NOs: 209-261); or combinations thereof.

In addition, the kits can include various diluents and buffers, labeled conjugates or other agents for the detection of specifically bound antigens or antibodies (e.g. labeling reagents), and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens. In some embodiments, the kit comprises an anti-human IgG/IgM antibody conjugated to a detectable label (e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex particle, or enzyme) as a labeling reagent. In other embodiments, the kit comprises protein A, protein G, protein A/G fusion proteins, protein L, or combinations thereof conjugated to a detectable label (e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex particle, or enzyme) as a labeling reagent. In still other embodiments, the labeling reagents of the kit are a second collection or set of peptides of the invention conjugated to a detectable label (e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex particle, or enzyme). The second collection or set of peptides can be the same as or different than the collection or set of peptides, which may optionally be attached to or immobilized upon a solid support.

Other components of a kit can easily be determined by one of skill in the art. Such components may include coating reagents, polyclonal or monoclonal capture antibodies specific for a peptide of the invention, or a cocktail of two or more of the antibodies, purified or semi-purified extracts of these antigens as standards, monoclonal antibody detector antibodies, an anti-mouse, anti-dog, anti-cat, anti-chicken, or anti-human antibody conjugated to a detectable label, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, a sample preparatory cup and buffers or other reagents appropriate for constituting a reaction medium allowing the formation of a peptide-antibody complex.

Such kits provide a convenient, efficient way for a clinical laboratory to diagnose infection by corona virus. Thus, in certain embodiments, the kits further comprise instructions.

EXAMPLES

The present invention may be better understood by reference to the following non-limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention.

Example 1—Materials and Methods Generation of Human Peptide Array-HCoV SeroChip

The platform for epitope discovery was a programmable peptide microarray that can accommodate up to 3 million distinct linear peptides on a 75 mm×26 mm slide (Nimblegen-Roche). The array can also be divided into 12 subarrays, each containing approximately 173,000 “12-mer peptides”. The 12-mer format was based on the observation that serum antibodies bind linear peptide sequences ranging from 5 to 9 amino acid (aa) and bind most efficiently when targets are flanked by additional amino acids (Buus et al. 2012). To enable differential detection of antibodies specific for SARS-CoV-2 infections, a custom NCBI GenBank and VIPR database for full proteomes of seven human coronaviruses: SARS-CoV-2; SARS; MERS; NL-63; OC-43; 229E; and HKU1, was created (Table 1). Two bat coronavirus proteomes similar to SARS-CoV-2 were also included (Zhou et al. 2020). 1000 randomly selected 12 amino acid long scrambled peptides were added for background correction and nonspecific binding of peptides.

For each virus selected, all available protein sequences available before January 2020 from the NCBI and Virus Pathogen Database and Analysis Resource (VIPR) protein databases were downloaded and then a peptide database created comprising overlapping 12-mer peptides that tiled the whole proteome of each of these agents with 11 amino acid (aa) overlap in a sliding window pattern (Tokarz et al. 2020; Tokarz et al. 2018; Mishra et al. 2019; Mishra et al. 2018). The selected peptide sequences were passed through a redundancy filter to yield 283,630 unique peptide sequences and 24,779,921 non-unique (present in more than one virus) for a total of 172,665 peptides. Redundant peptides were excluded prior to synthesis. The individual peptides in the library were printed in random positions on the peptide array to minimize the impact of locational bias.

TABLE 1 Design and Characteristics of HCoV-peptide Array Total Total No. of redundant unique protein “12-aa” “12-aa” Virus database sequences peptides peptides 1 SARS-CoV-2-MN908947-CDS 10 9591 9591 2 Human-229E-CoV-PDB 662 576999 15756 3 Human-HKU1-CoV-PDB 721 554447 16390 4 Human-NC63-CoV-PDB 1320 1000287 16392 5 Human-OC43-CoV-PDB 2667 2679272 29635 6 MERS-CoV-PDB 7253 8604059 54188 7 SARS-CoV-PDB 1996 1484140 29791 8 Human-CoV-variants-VIPR-PDB 12131 9880717 101145 9 Bat-CoV-ZC45 11 9678 9678 10 Bat-CoV-ZXC21 11 9655 9655 11 Redundant-non-specific-peptides NA NA 1000 Total number of unique and redundant peptides 24779921 283630 Total peptides in HCOV-SeroChip 172665

Samples and Experimental Design

The study was approved by the Medical Ethical Committee of Sun Yat-Sen University (approval number 2020-060). An informed and written consent was obtained by patients.

A total 132 plasma samples were tested and analyzed using HCoV peptide arrays. Samples were divided into six groups: group 1] COVID-19 patients with non-severe (mild) disease (n=22); group 2] COVID-19 patients with severe disease (n=22); group 3] patients with SARS-CoV-2 infections but no-symptoms (asymptomatic COVID-19) (n=6); group 4] SARS-2003 IgG positive cases (n=11); group 5] other banked HCoV IgG positive controls (n=11); and group 6] healthy controls (n=10). The average age was 44.0±16.73 years for the mild COVID-19 group, 60.1±12.37 years for the severe COVID-19 group, and 43.5±15.08 years for the asymptomatic COVID-19 group. The asymptomatic group subjects were family members or close contacts of the mild or severely ill patients. The average age for SARS-CoV-1 IgG positive control group was 24.4±5.0 years. The average age for healthy control group was 46.3±7.3 years. Other HCoV controls included banked samples for which age and sex information was not available.

Plasma samples were collected at two different time points, a minimum of two weeks apart, from group COVID-19 patients (groups 1, 2 and 3). The first time point (early) was collected at 12.9±5.9 post onset of disease (POD) for the mild disease group, and at 9.6±3.5 days POD for the severe disease group. The second time point (late) was collected at 34.7±8.3 days POD for the mild disease group, and at 24.8±6.8 days POD for the severe disease group. For asymptomatic group, the first time point was collected on the day of hospitalization and the second time point was collected at 14.5±4.6 days after the day of hospitalization. Non COVID-19 samples from control groups (group 4, group 5 and group 6), were from adults without any evidence or history of infection with SARS-CoV-2.

All COVID-19 patients were tested for SARS-CoV-2 RNA in respiratory specimens using the China FDA approved Novel Coronavirus (2019-nCoV) Real Time RT-PCR kit from LifeRiver Ltd. (Catalog #: RR-0479-02) real-time RT-PCR (CDC 2003). The diagnosis of COVID-19 pneumonia, and severity criteria were assessed at Guangdong CDC based on the New Coronavirus Pneumonia Prevention and Control Program (6th edition) published by the National Health Commission of China (Wu et al. 2020).

HCOV Peptide Array Synthesis, Sample Binding and Processing

Peptide synthesis was accomplished by light-directed array synthesis in a Nimble therapeutics Maskless Array Synthesizer (MAS) using an amino-functionalized substrate coupled with 6-amino hexanoic acid as a spacer and amino acid derivatives carrying a photosensitive 2-(2-Nitrophenyl) propyl-oxy-carbonyl-group (NPPOC) protection group (Orgentis Chemicals) Amino acids (final concentration 20 mM) were pre-mixed for 10 minutes in N,N-Dimethylformamide (DMF, Sigma Aldrich) with N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl) uroniumhexafluorophosphate (HBTU, Protein Technologies, Inc.; final concentration 20 mM) as an activator, 6-Chloro-1-hydroxybenzotriazole (6-Cl-HOBt, Protein Technologies, Inc.; final concentration 20 mM) to suppress racemization, and N,N-Diisopropylethylamine (DIPEA, Sigma Aldrich; final concentration 31 mM) as a base. Activated amino acids were then coupled to the array surface for 3 minutes. Following each coupling step, the microarray was washed with N-methyl-2-pyrrolidone (VWR International), and site-specific cleavage of the NPPOC protection group was accomplished by irradiation of an image created by a Digital Micro-Mirror Device (Texas Instruments), projecting 365 nm wavelength light. Coupling cycles were repeated to synthesize the full in silico-generated peptide library.

Eleven arrays were used to test 132 plasma samples. Before loading, plasma samples were heat inactivated at 56° C. for 30 minutes. Plasma samples were diluted (1:50) with binding buffer (0.1M Tris-Cl, 1% alkali soluble casein, 0.05% Tween-20, and water). The peptide arrays were incubated overnight at 4° C. on a flat surface with individual sample/sub-array. Overnight sample incubation was followed by three 10-minute washes with 1×TBS-T (0.05% Tween-20) at room temperature (RT). Secondary antibodies IgG (cat no. 109-605-098, Alexa Fluor 647-AffiniPure Goat Anti-Human IgG, Fcy fragment specific, Jackson ImmunoResearch Labs) and IgM (cat no. 109-165-129, Cy™3 AffiniPure Goat Anti-Human IgM, Jackson ImmunoResearch Labs) were diluted in 1×PBS at a concentration of 0.1 μg/ml, and arrays were incubated in Plastic Coplin Jar (cat no. 590130, Fisher Scientific) for 3 hours at RT with gentle shaking. Secondary antibody incubation was also followed by three 10-minute washes with 1×TBST at RT. After a final wash, the arrays were dried and scanned on a microarray scanner at 2-μm resolution, with an excitation wavelength of 635 nm (IgG) and 532 nm (IgM). The images were analyzed using the PepArray analysis program. The fluorescent signals were converted into arbitrary unit (AU) intensity plots ranging minimum to maximum intensity 0-65,000 AU.

Quality and Reproducibility of the Array Data

In the array synthesis process, Nimble Therapeutics uses a quality control step that builds thousands of peptides with known binding epitopes to streptavidin, which have been confirmed through SPR and crystallization method (Lyamichev et al. 2017). Each subarray on the entire synthesized array slide contained these quality control peptides in addition to the customized targeted experimental peptides. Additionally, every array slide was QC analyzed and the signal AU from these control peptides for each slide was correlated to the banked data from previous QC analyses. Arrays that did not meet standard cutoff thresholds were deemed as failed and removed from the further experimental process. Random nonadjacent tiling of overlapping peptides also enhanced confidence in the probe quality and supported the reactivity of an individual epitope by having multiple, overlapping peptides per epitope. In our previous studies, epitope reactivity of 12 aa overlapping peptides with an 11 amino acid overlap (single aa tiling) has been shown.

The reproducibility of data and inter-array reproducibility analysis of peptide arrays have been reported in previous studies by testing technical replicates on two separate microarrays. The two technical replicates for the target epitopes showed almost identical results with distinct epitopes (Tokarz et al. 2018; Mishra et al. 2018; Forsstrom et al. 2014).

All analysis similar to IgG was also performed to generate data from IgM reactive peptides. For IgM analysis, >10,000 AU filtration step reduced the initial number of peptides from 172,665 to 24,728 for further analysis. A total of 10,816 peptides (7144 from the COVID-19 group and 3672 from the control group) peptides yielded group-specific differences (p<0.05) in signal intensity. MDS efforts did not separate samples collected from patients with COVID-19 and controls (FIG. 1 ). However, the presence of three continuous peptides that were reactive in samples from SARS-CoV-2-infected subjects (irrespective of disease status) but not in control groups, allowed the identification of 16 SARS-CoV-2-specific IgM epitopes (Table 3).

Statistics and Reproducibility

Reactivity values for all 132 samples were pooled together and peptides showing >10,000 AU for any samples were retained for further analysis. Regression analysis, fold-changes, and standard errors were estimated by fitting a linear model for signal intensities generated by each peptide, applying empirical Bayesian smoothing to the standard errors, and then determining those peptides that yielded statistically significant signal by contrasting linear models for each peptide between SARS-CoV-2 and Control samples at a significance value of <0.0520. edgeR package in R was used for this purpose. It implements quantile-adjusted conditional maximum-likelihood method for estimating dispersions followed by fitting negative binomial generalized linear models. This was followed by a quasi-likelihood F-test to determine those peptides that yielded statistically significant signal by contrasting linear models for each peptide between SARS-CoV-2 and Control samples. MDS plots (FIG. 2 ) shows the discriminative ability of these peptides. These peptides were then used to reassemble longer epitope sequences (Table 2, Table 3). The normalization, background correction was performed using preprocessCore R package (Bolstad et al. 2003) and statistical comparison of peptide microarray intensities between groups were performed using the edgeR package (Robinson et al. 2010).

The analysis was performed to differentiate peptides that were immunoreactive with COVID-19 patients (groups 1, 2, and 3) and versus control groups (groups 4, 5, and 6) samples. MDS plots were generated using signal data for these significant peptides. The code for reassembly and plots was prepared using Rstudio v 1.2.5019. The plots were generated using ggplot2 package. A custom color-blind friendly color pallete was used to make the plots. Alignment of reactive epitopes on SARS-CoV-2 proteome was performed using Geneious version 10.0.9.

Example 2—Results

As described in Example 1, plasma samples were heat-inactivated at 56° C. for 30 minutes, diluted (1:50), added to HCoV peptide arrays, incubated with Alexa Fluor 647 labeled goat anti-human IgG, Cy™3 labeled goat anti-human IgM antibodies, and scanned on a microarray scanner.

Fluorescence signal data for all the peptides from IgG and IgM scanned images of all HCoV peptide arrays was converted to arbitrary units (AU), pooled, background corrected, and normalized to avoid any inter-experimental variations (Tokarz et al. 2018; Mishra et al. 2018; Buus et al. 2012). A peptide signal was considered reactive if the intensity reading (AU) was above the threshold (mean±2 SD readings of random peptides, >10,000 AU for IgG and IgM analysis). A cutoff threshold for peptide recognition was defined as mean ±2 times the standard deviation of the mean intensity value of all negative controls (Valentini et al. 2017). The regression analysis was performed to measure fold-changes on normalized and background corrected data for filtered peptides and followed by multidimensional scaling (MDS) analysis.

For IgG analysis, the >10,000 AU filtration step reduced the initial number of peptides from 172,665 to 79,714 for further analysis. A total of 37,237 peptides (18,533 from the COVID-19 group and 18,704 from the control group) were identified by regression analysis. This yielded group specific differences (p<0.05) in signal intensity. MDS analysis was performed for IgG and IgM antibodies to differentiate peptides that were immunoreactive with COVID-19 patients (Groups 1-3) versus control groups (Group 4-6). MDS analysis of these peptides confirmed separation of patients with COVID-19 (Groups 1-3) and controls (Groups 4-6) into separate clusters with minimal overlap (FIG. 2 ). Out of 18,533 reactive 12-mer peptides associated with the COVID-19 group, 981 peptides were specific for the SARS-CoV-2 polyprotein.

The 981 SARS-CoV-2 peptides with IgG signal intensity >10,000 AU, were used to assemble a heatmap. The signal data was pooled from all the arrays and then reactivity compared across different sample groups. The pooled data were normalized; background corrected, and filtered peptides with immunoreactivity above the 10,000AU to generate the heatmap shown in FIG. 3 . Immunoreactive peptides included 566 from ORF1ab, three from ORF10, 243 from surface glycoprotein (S protein), 20 from ORF3a, 20 from membrane glycoprotein (mGP), four from ORF7a, 21 from ORFS, and 104 from nucleocapsid phosphoprotein (N). Peptides from “S” and “N” proteins had higher reactive intensity (higher AU) and rate of reactivity in comparison to peptides from other proteins (FIG. 3 ). Immunoreactivity was higher in COVID-19 subjects with severe versus mild disease, and in asymptomatic SARS-CoV-2-infected subjects versus subjects with mild disease. Samples from second time point collections were more reactive than first time point collections in subjects with severe, mild and asymptomatic SARSCoV-2 infection (FIG. 3 ).

The presence of a minimum of three continuous reactive peptides in samples with SARS-CoV-2 infection but non-reactive in control groups, were used to identify SARS-CoV-2 reactive IgG epitopes. Analysis of these 981 peptide sequences led to the identification of 163 epitopes (Table 4). The 29 epitopes with the strongest and most specific reactivity with SARS-CoV-2 are shown in Table 2. Table 2 also indicates the location of each epitope on the SARS-CoV-2 proteome, its length, aa sequence, and the percentages of plasma samples that were immunoreactive in Groups 1-6.

These 29 epitopes included 11 epitopes (37.9%) in S protein (SP1-SP11), 8 (27.5%) epitopes in N protein (NP1-NP8), 6 (20.7%) epitopes in ORF1ab polyprotein (OP1-OP6), 2 (6.9%) in mGP protein (MP1 and MP2), one (3.4%) each from ORF3, and ORF8 proteins. Immunoreactivity was higher in second time point plasma samples (FIG. 4 ) and in patients with more severe disease. In samples from patients with severe disease, 7-22 epitopes (mean of 13) were reactive in second time point samples (24.8±6.8 days POD) versus 0-15 (mean of 7) epitopes in first time point samples (9.6±3.5 days POD). In patients with mild disease, 3-22 epitopes (mean of eight) were reactive in second time point samples (34.7±8.3 POD) versus 0-12 epitopes (mean of 4) in first time point samples (12.9±5.9 POD). In asymptomatic subjects 1-9 epitopes (mean of four) were reactive at either time point (day of hospitalization and 14.5±4.6 days after the hospitalization). Plasma samples from 19 of 22 patients (86%) with mild disease were reactive with at least 1 of 29 epitopes at the first time point. All 22 (100%) were reactive with at least 3 of 29 epitopes at the second time point. Plasma from 21 of 22 patients (95%) with severe disease were reactive with at least one epitope at first time point. All 22 (100%) were reactive with at least six epitopes at the second time point. All six (100%) asymptomatic cases were reactive with at least 2 of 29 epitopes in first and 3 of 29 in second timepoint collections.

Plasma from none of the asymptomatic cases was reactive with any ORF1ab, ORF3, ORF8, or mGP epitope (Table 4). Plasma from asymptomatic cases were reactive only with S and N epitopes. Plasma collected at both time points from asymptomatic case AS1 was reactive with five epitopes in the S protein (SP1, SP2, SP4, SP5, and SP10) and three epitopes in the N protein (NP2, NP7, and NP8). Case AS1 plasma was reactive with SP9 at only first time point. Plasma from asymptomatic case AS2 was reactive with three epitopes in the S protein (SP7, SP8, and SP10) and five epitopes in the N protein (NP2, NP3, NP4, NPS, and NP8) at both time points. Plasma from asymptomatic cases AS1, AS2, and AS4 were reactive with SP10 and NP8 epitopes at both time points. Plasma from AS3 was reactive with SP10 at both time points and with NP8 only at the second time point. Plasma from AS5 and AS6 were immunoreactive with the NP1 epitope and the epitope NP6, respectively, at both time points. Plasma from mild case M7 was reactive only with the SP10 epitope at first time point but was reactive to 22/29 epitopes at second time point of collection. All mild and severe COVID-19 cases were reactive to a greater number of epitopes at second time points versus the first time point.

The 29 immunoreactive epitopes were mapped to the proteome of SARS-CoV-2 (acc. number MN908947). ORF1ab epitopes (OP1-OP6) were dispersed throughout the protein. Eleven linear epitopes from “S” protein (SP1-SP11) mapped outside of the RBD (Receptor Binding Domain). Four (SP1-SP4) epitopes were located in the SD1/SD2. Four (SP5-SP8) epitopes were located between SD1/SD2 and fusion peptide region. The SP9 epitope overlapped the fusion peptide. The SP10 epitope is located between CD (Connector Domain) region and HR2 (Heptad Repeat 2). The SP11 epitope was located at the beginning of the HR2 region of the S2 subunit of the surface glycoprotein. The SP1 and SP9 peptides identified in this study were recently reported as potentially linked to neutralization (Poh et al. 2020).

Ten of eleven spike epitopes (SP1-SP10) were located in regions of high immunoreactivity reported in a recent pre-print from Li et al. 2020. The SP10 epitope was the most reactive spike epitope in subjects with severe (69% first time point, 96% second timepoint), mild (41% first timepoint, 82% second timepoint), and asymptomatic SARS-CoV-2 (67% either timepoint) infections.

The NP2 and NP8 epitopes were the most reactive N protein epitopes. In severe disease, NP2 reactivity was found in 55% of subjects at the first timepoint, and 82% at the second timepoint. NP8 reactivity was found in 64% of subjects at first timepoint, and 100% at second timepoint. In mild disease, NP2 reactivity was found in 37% of subjects at the first timepoint, and 69% at the second timepoint. NP8 reactivity was found in 28% of subjects at the first timepoint, and 82% at the second timepoint. In asymptomatic cases, reactivity was 59% for either NP2 or NP8. In severe disease SP11 reactivity was found in 46% of subjects at the first time point, and 91% at the second time point. In mild disease SP11 reactivity was found in 28% of subjects at the first time point, and 60% at the second time point. No asymptomatic cases were reactive with SP11. Epitopes for ORF1ab (OP1-OP6) showed reactivity in cases with mild and severe disease at both timepoints but not in asymptomatic infections. Only second timepoint samples from severe and mild diseases were reactive to epitopes for mGP, MP1, and MP2 (37 and 26% for severe disease and 28 and 19% for mild disease).

IgM analysis revealed one linear epitope in the membrane glycoprotein (MADSNGTITVEELKKLLEQWN) (SEQ ID NO: 42), that was reactive in 4/22 (19%) COVID-19 patients with mild disease and 8/22 (37%) patients with severe disease at late time point collection (FIG. 1 , Table 3).

Through use of a limited number of plasma samples from subjects with known exposure to other HCoV, IgG epitopes were identified in HKU1 (n=15), NL63 (n=10), OC43 (n=14), 229E (n=5), and SARS-CoV-1 (n=9) (Table 5). More than 90% of samples from patients exposed to SARS-CoV-2 and controls showed a wide range of reactivity to epitopes from seasonal coronaviruses (HKU1, NL63, OC43, and 229E).

In summary, the HCoV peptide array and plasma from 50 patients with asymptomatic, mild, or severe SARS-CoV-2 infection was used to identify immunoreactive IgG epitopes for SARSCoV-2. Immunoreactivity profiles differed with severity of illness and over the time course of infection. Two subjects with a history of SARS-CoV-1 infection had reactivity to two of 29 IgG SARSCoV-2 epitopes. Their plasma was collected in 2004 or 2005; thus, this presumably reflects cross-reactivity due to proteome homology (Lv et al. 2020; Chia et al. 2020). Two healthy controls with immunoreactivity to SP11 (one of 29 epitopes of SARS-CoV-2) may have had an asymptomatic infection with either SARS-CoV-1 or SARS-CoV-2 (Xu et al. 2006; CDC 2003).

TABLE 2 IgG linear epitopes for detection of SARS-CoV-2 infection Epitope AA AA Protein name S. No. Epitope ID. Epitope Sequence length (start) (end) (Acc. MN908947) 1 OP1 TAYNGYLTSSSKTP (SEQ ID NO: 1) 13 1,484 1,497 ORF1ab 2 OP2 LVPNQPYPNASFDNF (SEQ ID NO: 2) 14 1,914 1,928 polyprotein 3 OP3 NRFTTTLNDFNLVAMK (SEQ ID NO: 3) 15 3,484 3,499 (pp1ab) 4 OP4 QGLLPPKNSIDAFKLNIK (SEQ ID NO: 4) 17 3,826 3,843 5 OP5 NTCVGSDNVTDFNAIATCDW (SEQ ID NO: 5) 19 5,419 5,438 6 OP6 FYSYATHSDKFTDGV (SEQ ID NO: 6) 14 6,292 6,306 7 SP1 TESNKKFEPFQQFGRDIAD (SEQ ID NO: 7) 18 553 571 Surface 8 SP2 TDAVRDPQTLEILDITPCS (SEQ ID NO: 8) 18 573 591 glycoprotein 9 SP3 QVAVLYQDVNCTEVPVAIHADQLTPTW (SEQ ID NO: 9) 26 607 633 10 SP4 CASYQTQTNSPRRARSV (SEQ ID NO: 10) 16 671 687 11 SP5 ARSVASQSIIAYTMSLGAENSVA (SEQ ID NO: 11) 22 684 706 12 SP6 RALTGIAVEQDKNTQEVFAQV (SEQ ID NO: 12) 20 765 785 13 SP7 FAQVKQIYKTPPIKDFGGFNF (SEQ ID NO: 13) 20 782 802 14 SP8 NFSQILPDPSKPSKRS (SEQ ID NO: 14) 15 801 816 15 SP9 KRSFIEDLLFNKVT (SEQ ID NO: 15) 13 814 827 16 SP10 PELDSFKEELDKYFKNHTSPDVD (SEQ ID NO: 16) 22 1,143 1,165 17 SP11 SPDVDLGDISGINASVV (SEQ ID NO: 17) 16 1,161 1,177 18 ORF3-1 EHDYQIGGYTEKWESGVKDCVVL (SEQ ID NO: 18) 22 181 203 ORF3a protein 19 MP1 CDIKDLPKEITVATS (SEQ ID NO: 19) 14 159 173 Membrane 20 MP2 QRVAGDSGFAAYSRY (SEQ ID NO: 20) 14 185 199 glycoprotein 21 ORF8-1 GSKSPIQYIDIGNYTVSCLPF (SEQ ID NO: 21) 20 66 86 ORF8 protein 22 NP1 ALLLLDRLNQLESKMSG (SEQ ID NO: 22) 16 220 236 Nucleocapsid 23 NP2 NQLESKMSGKGQQQQGQTVTKKSA (SEQ ID NO: 23) 23 228 251 phopsphoprotein 24 NP3 QTVTKKSAAEASKKPRQK (SEQ ID NO: 24) 17 244 261 25 NP4 RGPEQTQGNFGDQELI (SEQ ID NO: 25) 15 277 292 26 NP5 AYKTFPPTEPKKDKKKK (SEQ ID NO: 26) 16 359 375 27 NP6 KDKKKKADETQALPQR (SEQ ID NO: 27) 15 370 385 28 NP7 ADETQALPQRQKKQQTVTLLPAADL (SEQ ID NO: 28) 24 376 400 29 NP8 TLLPAADLDDFSKQLQQSMSSADS (SEQ ID NO: 29) 23 393 416 Severe- Severe- Mild- Mild- Asymptomatic COVID- COVID- COVID- COVID- COVID- SARS- Healthy- Other- 19 Early 19 Late 19 Early 19 Late 19 Late CoV-1 Controls HCoV S. No. (N = 22) (N = 22) (N = 22) (N = 22) (N = 12) (N = 11) (N = 10) (N = 11) 1 5/22 (23%) 4/22 (19%) 5/22 (23%) 5/22 (23%) 0/12 (0%)   1/11 (10%) 0/10 (0%)  1/11 (10%) 2 8/22 (37%) 12/22 (55%)  3/22 (14%) 3/22 (14%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 3 1/22 (5%)  1/22 (5%)  4/22 (19%) 3/22 (14%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 4 5/22 (23%) 5/22 (23%) 4/22 (19%) 6/22 (28%) 0/12 (0%)  0/11 (0%) 0/10 (0%)  2/11 (19%) 5 5/22 (23%) 13/22 (60%)  5/22 (23%) 6/22 (28%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 6 7/22 (32%) 11/22 (50%)  0/22 (0%)  2/22 (10%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 7 4/22 (19%) 15/22 (69%)  1/22 (5%)  9/22 (41%) 3/12 (25%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 8 6/22 (28%) 17/22 (78%)  1/22 (5%)  11/22 (50%)  2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 9 0/22 (0%)  9/22 (41%) 2/22 (10%) 4/22 (19%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 10 2/22 (10%) 4/22 (19%) 0/22 (0%)  1/22 (5%)  2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 11 2/22 (10%) 6/22 (28%) 4/22 (19%) 4/22 (19%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 12 11/22 (50%)  14/22 (64%)  2/22 (10%) 5/22 (23%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 13 5/22 (23%) 8/22 (37%) 1/22 (5%)  3/22 (14%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 14 1/22 (5%)  4/22 (19%) 0/22 (0%)  4/22 (19%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 15 5/22 (23%) 8/22 (37%) 4/22 (19%) 5/22 (23%) 1/12 (9%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 16 15/22 (69%)  21/22 (96%)  9/22 (41%) 18/22 (82%)  8/12 (67%)  1/11 (10%) 0/10 (0%)  1/11 (10%) 17 10/22 (46%)  20/22 (91%)  6/22 (28%) 13/22 (60%)  0/12 (0%)   1/11 (10%)  2/10 (20%)  1/11 (10%) 18 4/22 (19%) 7/22 (32%) 1/22 (5%)  2/22 (10%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 19 0/22 (0%)  8/22 (37%) 0/22 (0%)  6/22 (28%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 20 0/22 (0%)  10/22 (46%)  0/22 (0%)  4/22 (19%) 0/12 (0%)  0/11 (0%) 0/10 (0%) 0/11 (0%) 21 3/22 (14%) 12/22 (55%)  4/22 (19%) 3/22 (14%) 0/12 (0%)  0/11 (0%) 0/10 (0%)  1/11 (10%) 22 3/22 (14%) 5/22 (23%) 2/22 (10%) 5/22 (23%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 23 12/22 (55%)  18/22 (82%)  8/22 (37%) 15/22 (69%)  7/12 (59%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 24 2/22 (10%) 7/22 (32%) 1/22 (5%)  4/22 (19%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 25 6/22 (28%) 3/22 (14%) 2/22 (10%) 3/22 (14%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 26 2/22 (10%) 5/22 (23%) 0/22 (0%)  2/22 (10%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 27 3/22 (14%) 7/22 (32%) 0/22 (0%)  5/22 (23%) 3/12 (25%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 28 4/22 (19%) 17/22 (78%)  4/22 (19%) 9/22 (41%) 2/12 (17%) 0/11 (0%) 0/10 (0%) 0/11 (0%) 29 14/22 (64%)  22/22 (100%) 6/22 (28%) 18/22 (82%)  7/12 (59%)  1/11 (10%) 0/10 (0%) 0/11 (0%)

TABLE 3 IgM linear epitopes for detection of SARS-CoV-2 infection Healthy- Mild- Mild- Controls_ COVID_ COVID_ Other_ Asymtomatic China Early Late CoV  1 LGDELGTDPYEDFQ SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 1/12 (9%) NO: 30  2 LYIDINGNLHPDSAT SEQ ID 0/12 (0%) 0/10 (0%) 2/22 (10%) 0/22 (0%) 0/12 (0%) NO: 31  3 ATKYLVQQESPFVMM SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 1/12 (9%) NO: 32  4 IVFDGKSKCEESSA SEQ ID 0/12 (0%) 0/10 (0%) 1/22 (5%) 1/22 (5%) 0/12 (0%) NO: 33  5 LNDFSNSGSDVLYQP SEQ ID 0/12 (0%) 1/10 (10%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 34  6 LSFCAFAVDAAKAY SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 35  7 YDFGDFIQTTPGSG SEQ ID 0/12 (0%) 1/10 (10%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 36  8 DFIQTTPGSGVPVV SEQ ID 0/12 (0%) 1/10 (10%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 37  9 FLLKYNENGTITDAV SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 38 10 GVLTESNKKFLPFQ SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 39 11 YQTQTNSPRRARSVA SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 40 12 NTQEVFAQVKQIYK SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 41 13 MADSNGTITVEELKKLLEQWN SEQ ID 1/12 (9%) 0/10 (0%) 0/22 (0%) 4/22 (19%) 0/12 (0%) NO: 42 14 KEPCSSGTYEGNSPFHP SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 1/22 (5%) 1/12 (9%) NO: 43 15 TLLPAADLDDFSKQ SEQ ID 1/12 (9%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 44 16 AADLDDFSKQLQQS SEQ ID 0/12 (0%) 0/10 (0%) 0/22 (0%) 0/22 (0%) 0/12 (0%) NO: 45 Severe- Severe- COVID_ SARS_2003 COVID_Early Late  1 LGDELGTDPYEDFQ SEQ ID NO: 30 0/10 (0%) 0/22 (0%) 0/22 (0%)  2 LYIDINGNLHPDSAT SEQ ID NO: 31 0/10 (0%) 0/22 (0%) 2/22 (10%)  3 ATKYLVQQESPFVMM SEQ ID NO: 32 0/10 (0%) 0/22 (0%) 0/22 (0%)  4 IVFDGKSKCEESSA SEQ ID NO: 33 0/10 (0%) 0/22 (0%) 0/22 (0%)  5 LNDFSNSGSDVLYQP SEQ ID NO: 34 0/10 (0%) 0/22 (0%) 0/22 (0%)  6 LSFCAFAVDAAKAY SEQ ID NO: 35 0/10 (0%) 1/22 (5%) 0/22 (0%)  7 YDFGDFIQTTPGSG SEQ ID NO: 36 0/10 (0%) 0/22 (0%) 0/22 (0%)  8 DFIQTTPGSGVPVV SEQ ID NO: 37 0/10 (0%) 0/22 (0%) 0/22 (0%)  9 FLLKYNENGTITDAV SEQ ID NO: 38 0/10 (0%) 0/22 (0%) 1/22 (5%) 10 GVLTESNKKFLPFQ SEQ ID NO: 39 1/10 (10%) 0/22 (0%) 0/22 (0%) 11 YQTQTNSPRRARSVA SEQ ID NO: 40 0/10 (0%) 0/22 (0%) 1/22 (5%) 12 NTQEVFAQVKQIYK SEQ ID NO: 41 0/10 (0%) 1/22 (5%) 0/22 (0%) 13 MADSNGTITVEELKKLLEQWN SEQ ID NO: 42 0/10 (0%) 1/22 (5%) 8/22 (37%) 14 KEPCSSGTYEGNSPFHP SEQ ID NO: 43 0/10 (0%) 0/22 (0%) 0/22 (0%) 15 TLLPAADLDDFSKQ SEQ ID NO: 44 0/10 (0%) 1/22 (5%) 1/22 (5%) 16 AADLDDFSKQLQQS SEQ ID NO: 45 0/10 (0%) 1/22 (5%) 0/22 (0%)

TABLE 4 Additional IgG linear epitopes for detection of SARS-CoV-2 infection.  Sequences (aa) are based on proteome of Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 (Accession no. MN908947) Protein Info Severe- Severe- Mild- Mild- (MN908947_CDS_ COVID_ COVID_ COVID_ COVID_ Epitope sequence proteome) Early Late Early Late YRKVLLRKNGNKGA SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 46 tein (pp1ab) (5%) (0%) (0%) LLRKNGNKGAGGHSY SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 47 tein (pp1ab) (0%) (0%) (0%) GNKGAGGHSYGADLKSF SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  DL NO: 48 tein (pp1ab) (0%) (0%) (0%) DELGTDPYEDFQEN SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 49 tein (pp1ab) (0%) (0%) (0%) WNTKHSSGVTRELM SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  NO: 50 tein (pp1ab) (0%) (5%) (5%) GVYCCREHEHEIAW SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 51 tein (pp1ab) (0%) (0%) (0%) HEIAWYTERSEKSY SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 52 tein (pp1ab) (0%) (0%) (0%) SYELQTPFEIKLAK SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 53 tein (pp1ab) (5%) (0%) (0%) AKKFDTFNGECPNF SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 54 tein (pp1ab) (0%) (0%) (0%) CDHCGETSWQTGDF SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  NO: 55 tein (pp1ab) (0%) (5%) (5%) LTNIFGTVYEKLKPVLD SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 56 tein (pp1ab) (0%) (0%) (0%) GVEFLRDGWEIVKF SEQ ID orf1ab polypro- 1/22 (5%) 0/22 0/22  0/22  NO: 57 tein (pp1ab) (0%) (0%) (0%) TGDLQPLEQPTSEAVEAP SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  LV NO: 58 tein (pp1ab) (0%) (0%) (0%) KGGAPTKVTFGDDTVIEV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 59 tein (pp1ab) (0%) (0%) (0%) NITFELDERIDKVL SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 60 tein (pp1ab) (5%) (0%) (0%) EFACVVADAVIKTLQPVS SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  1/22  NO: 61 tein (pp1ab) (5%) (0%) (5%) NKGEDIQLLKSAYENF SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 62 tein (pp1ab) (5%) (0%) (0%) YDKLVSSFLEMKSEK SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 63 tein (pp1ab) (5%) (0%) (0%) AFYILPSIISNEKQEI SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 64 tein (pp1ab) (5%) (0%) (0%) LGYVTHGLNLEEAA SEQ ID orf1ab polypro- 1/22 (5%) 2/22 0/22  0/22  NO: 65 tein (pp1ab) (10%) (0%) (0%) THGLNLEEAARYMRS SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  1/22  NO: 66 tein (pp1ab) (5%) (0%) (5%) TAYNGYLTSSSKTP SEQ ID orf1ab polypro- 5/22 (23%) 4/22 5/22 5/22 NO: 67 tein (pp1ab) (19%) (23%) (23%) GEVITFDNLKTLLS SEQ ID orf1ab polypro- 0/22 (0%) 1/22 1/22  1/22  NO: 68 tein (pp1ab) (5%) (5%) (5%) GRYMSALNHTKKWKYPQ SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  V NO: 69 tein (pp1ab) (5%) (0%) (0%) TVGELGDVRETMSYL SEQ ID orf1ab polypro- 1/22 (5%) 0/22 1/22  0/22  NO: 70 tein (pp1ab) (0%) (5%) (0%) MGTLSYEQFKKGVQIPC SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 71 tein (pp1ab) (5%) (0%) (0%) FTCASEYTGNYQCGHY SEQ ID orf1ab polypro- 2/22 (10%) 2/22 3/22 3/22 NO: 72 tein (pp1ab) (10%) (14%) (14%) YQCGHYKHITSKET SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 73 tein (pp1ab) (0%) (0%) (0%) LVPNQPYPNASFDNF SEQ ID orf1ab polypro- 8/22 (37%) 12/22 3/22 3/22 NO: 74 tein (pp1ab) (55%) (14%) (14%) FPDLNGDVVAIDYK SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 75 tein (pp1ab) (5%) (0%) (0%) NSFDVLKSEDAQGMDNL SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 76 tein (pp1ab) (0%) (0%) (0%) SEEVVENPTIQKDVLECN SEQ ID orf1ab polypro- 0/22 (0%) 2/22 0/22  1/22  NO: 77 tein (pp1ab) (10%) (0%) (5%) GLAAVNSVPWDTIA SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  0/22  NO: 78 tein (pp1ab) (0%) (5%) (0%) TIANYAKPFLNKVVSTTT SEQ ID orf1ab polypro- 1/22 (5%) 2/22 1/22  1/22  NO: 79 tein (pp1ab) (10%) (5%) (5%) FLNKVVSTTTNIVTRC SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 80 tein (pp1ab) (0%) (0%) (0%) VKMFDAYVNTFSST SEQ ID orf1ab polypro- 2/22 (10%) 3/22 1/22  0/22  NO: 81 tein (pp1ab) (14%) (5%) (0%) FSSTFNVPMEKLKT SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 82 tein (pp1ab) (0%) (0%) (0%) TLVATAEAELAKNVSL SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 83 tein (pp1ab) (0%) (0%) (0%) SAAKKNNLPFKLTCA SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 84 tein (pp1ab) (5%) (0%) (0%) VHVMSKHTDFSSEIIG SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 85 tein (pp1ab) (5%) (0%) (0%) DFDTWFSQRGGSYT SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 86 tein (pp1ab) (0%) (0%) (0%) TFDSEYCRHGTCER SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 87 tein (pp1ab) (0%) (0%) (0%) DVLYQPPQTSITSAVL SEQ ID orf1ab polypro- 2/22 (10%) 1/22 1/22  0/22  NO: 88 tein (pp1ab) (5%) (5%) (0%) NRFTTTLNDFNLVAMK SEQ ID orf1ab polypro- 1/22 (5%) 1/22 4/22 3/22 NO: 89 tein (pp1ab) (5%) (19%) (14%) LTQDHVDILGPLSA SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  1/22  NO: 90 tein (pp1ab) (0%) (0%) (5%) GMNGRTILGSALLED SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  1/22  NO: 91 tein (pp1ab) (0%) (0%) (5%) QGLLPPKNSIDAFKLNIK SEQ ID orf1ab polypro- 5/22 (23%) 5/22 4/22 6/22 NO: 92 tein (pp1ab) (23%) (19%) (28%) KLKKSLNVAKSEFDRD SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 93 tein (pp1ab) (0%) (0%) (0%) LRANSAVKLQNNELS SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 94 tein (pp1ab) (0%) (0%) (0%) AVKLQNNELSPVALR SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 95 tein (pp1ab) (5%) (0%) (0%) CAAGTTQTACTDDN SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  NO: 96 tein (pp1ab) (0%) (5%) (5%) LALLSDLQDLKWARF SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 97 tein (pp1ab) (5%) (0%) (0%) ATVRLQAGNATEVPA SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 98 tein (pp1ab) (0%) (0%) (0%) VKMLCTHTGTGQAITV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 2/22 2/22 NO: 99 tein (pp1ab) (0%) (10%) (10%) KYVQIPTTCANDPV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 100 tein (pp1ab) (0%) (0%) (0%) RVCGVSAARLTPCGT SEQ ID orf1ab polypro- 2/22 (10%) 1/22 0/22  0/22  NO: 101 tein (pp1ab) (5%) (0%) (0%) AVAKHDFFKFRIDG SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 102 tein (pp1ab) (0%) (0%) (0%) GDFIQTTPGSGVPV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 103 tein (pp1ab) (0%) (0%) (0%) QTYHPNCVNCLDDR SEQ ID orf1ab polypro- 0/22 (0%) 1/22 1/22  2/22 NO: 104 tein (pp1ab) (5%) (5%) (10%) ALTNNVAFQTVKPGNF SEQ ID orf1ab polypro- 1/22 (5%) 0/22 0/22  0/22  NO: 105 tein (pp1ab) (0%) (0%) (0%) GFFKEGSSVELKHF SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 106 tein (pp1ab) (5%) (0%) (0%) QLLFVVEVVDKYFDCYD SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  GGC NO: 107 tein (pp1ab) (0%) (5%) (5%) SLYVKPGGTSSGDATTAY SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  A NO: 108 tein (pp1ab) (0%) (0%) (0%) NRDVDTDFVNEFYAYL SEQ ID orf1ab polypro- 0/22 (0%) 1/22 2/22 2/22 NO: 109 tein (pp1ab) (5%) (10%) (10%) SQGLVASIKNFKSV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 110 tein (pp1ab) (0%) (0%) (0%) QHTMLVKQGDDYVY SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 111 tein (pp1ab) (5%) (0%) (0%) VDDIVKTDGTLMIE SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 112 tein (pp1ab) (5%) (0%) (0%) AIDAYPLTKHPNQE SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 113 tein (pp1ab) (0%) (0%) (0%) GCDVTDVTQLYLGG SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 114 tein (pp1ab) (5%) (0%) (0%) GLYKNTCVGSDNVT SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 115 tein (pp1ab) (5%) (0%) (0%) NTCVGSDNVTDFNAIATC SEQ ID orf1ab polypro- 5/22 (23%) 13/22 5/22 6/22 DW NO: 116 tein (pp1ab) (60%) (23%) (28%) YQKVGMQKYSTLQG SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 117 tein (pp1ab) (0%) (0%) (0%) STLQGPPGTGKSHF SEQ ID orf1ab polypro- 2/22 (10%) 0/22 1/22  2/22 NO: 118 tein (pp1ab) (0%) (5%) (10%) ARARVECFDKFKVNS SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  NO: 119 tein (pp1ab) (0%) (5%) (5%) DPAQLPAPRTLLTK SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 120 tein (pp1ab) (0%) (0%) (0%) PRTLLTKGTLEPEY SEQ ID orf1ab polypro- 2/22 (10%) 2/22 0/22  0/22  NO: 121 tein (pp1ab) (10%) (0%) (0%) IFTQTTETAHSCNV SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  1/22  NO: 122 tein (pp1ab) (5%) (0%) (5%) GCHATREAVGTNLP SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  NO: 123 tein (pp1ab) (0%) (5%) (5%) VQMLSDTLKNLSDR SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 124 tein (pp1ab) (5%) (0%) (0%) FDYVYNPFMIDVQQWG SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 125 tein (pp1ab) (0%) (0%) (0%) MIDVQQWGFTGNLQ SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 126 tein (pp1ab) (5%) (0%) (0%) TGNLQSNHDLYCQVH SEQ ID orf1ab polypro- 0/22 (0%) 1/22 0/22  0/22  NO: 127 tein (pp1ab) (5%) (0%) (0%) LADKFPVLHDIGNPKAIK SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  CVP NO: 128 tein (pp1ab) (0%) (0%) (0%) FYSYATHSDKFTDGV SEQ ID orf1ab polypro- 7/22 (32%) 11/22 0/22  2/22 NO: 129 tein (pp1ab) (50%) (0%) (10%) FDTRVLSNLNLPGCD SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 130 tein (pp1ab) (0%) (0%) (0%) QVVSDIDYVPLKSATC SEQ ID orf1ab polypro- 3/22 (14%) 3/22 1/22  1/22  NO: 131 tein (pp1ab) (14%) (5%) (5%) PSVGPKQASLNGVTL SEQ ID orf1ab polypro- 1/22 (5%) 0/22 0/22  0/22  NO: 132 tein (pp1ab) (0%) (0%) (0%) GVVQQLPETYFTQSRN SEQ ID orf1ab polypro- 1/22 (5%) 1/22 0/22  0/22  NO: 133 tein (pp1ab) (5%) (0%) (0%) AMDEFIERYKLEGY SEQ ID orf1ab polypro- 3/22 (14%) 6/22 0/22  1/22  NO: 134 tein (pp1ab) (28%) (0%) (5%) LAKRFKESPFELED SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 135 tein (pp1ab) (0%) (0%) (0%) FITDAQTGSSKCVCSVID SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 136 tein (pp1ab) (0%) (0%) (0%) IKSQDLSVVSKVVKV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 137 tein (pp1ab) (0%) (0%) (0%) YPKLQSSQAWQPGV SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  0/22  NO: 138 tein (pp1ab) (0%) (5%) (0%) QRMLLEKCDLQNYG SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 139 tein (pp1ab) (0%) (0%) (0%) HFGAGSDKGVAPGTA SEQ ID orf1ab polypro- 0/22 (0%) 0/22 0/22  0/22  NO: 140 tein (pp1ab) (0%) (0%) (0%) ITEHSWNADLYKLM SEQ ID orf1ab polypro- 0/22 (0%) 0/22 1/22  1/22  NO: 141 tein (pp1ab) (0%) (5%) (5%) LSSYSLFDMSKFPLKL SEQ ID orf1ab polypro- 2/22 (10%) 3/22 0/22  0/22  NO: 142 tein (pp1ab) (14%) (0%) (0%) SLLLCRMNSRNYIA SEQ ID ORF10 protein 1/22 (5%) 1/22 0/22  0/22  NO: 143 (5%) (0%) (0%) QCVNLTTRTQLPPA SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 144 glycoprotein (0%) (0%) (0%) TQLPPAYTNSFTRGVYYP SEQ ID surface  4/22 (19%) 5/22 2/22 2/22 D NO: 145 glycoprotein (23%) (10%) (10%) FHAIHVSGTNGTKRF SEQ ID surface  0/22 (0%) 0/22 1/22  0/22  NO: 146 glycoprotein (0%) (5%) (0%) NLVRDLPQGFSALEPLV SEQ ID surface  0/22 (0%) 1/22 1/22  1/22  NO: 147 glycoprotein (5%) (5%) (5%) LALHRSYLTPGDSSSGWT SEQ ID surface  1/22 (5%) 3/22 0/22  2/22 NO: 148 glycoprotein (14%) (0%) (10%) KYNENGTITDAVDCALDP SEQ ID surface  1/22 (5%) 1/22 1/22  1/22  LS NO: 149 glycoprotein (5%) (5%) (5%) IYQAGSTPCNGVEGFNCY SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 150 glycoprotein (0%) (0%) (0%) VLSFELLHAPATVC SEQ ID surface  1/22 (5%) 0/22 0/22  0/22  NO: 151 glycoprotein (0%) (0%) (0%) LLHAPATVCGPKKST SEQ ID surface  0/22 (0%) 2/22 0/22  1/22  NO: 152 glycoprotein (10%) (0%) (5%) VKNKCVNFNFNGLT SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 153 glycoprotein (0%) (0%) (0%) TESNKKFLPFQQFGRDIA SEQ ID surface  4/22 (19%) 15/22 1/22  9/22 D NO: 154 glycoprotein (69%) (5%) (41%) TDAVRDPQTLEILDITPC SEQ ID surface  6/22 (28%) 17/22 1/22  11/22 S NO: 155 glycoprotein (78%) (5%) (50%) QVAVLYQDVNCTEVPVAI SEQ ID surface  0/22 (0%) 9/22 2/22 4/22 HADQLTPTW NO: 156 glycoprotein (41%) (10%) (19%) IHADQLTPTWRVYSTGSN SEQ ID surface  0/22 (0%) 3/22 0/22  2/22 NO: 157 glycoprotein (14%) (0%) (10%) YSTGSNVFQTRAGCL SEQ ID surface  2/22 (10%) 1/22 0/22  0/22  NO: 158 glycoprotein (5%) (0%) (0%) CASYQTQTNSPRRARSV SEQ ID surface  2/22 (10%) 4/22 0/22  1/22  NO: 159 glycoprotein (19%) (0%) (5%) ARSVASQSIIAYTMSLGA SEQ ID surface  2/22 (10%) 6/22 4/22 4/22 ENSVA NO: 160 glycoprotein (28%) (19%) (19%) PVSMTKTSVDCTMY SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 161 glycoprotein (0%) (0%) (0%) RALTGIAVEQDKNTQEVF SEQ ID surface  11/22 (50%) 14/22 2/22 5/22 AQV NO: 162 glycoprotein (64%) (10%) (23%) FAQVKQIYKTPPIKDFGG SEQ ID surface  5/22 (23%) 8/22 1/22  3/22 FNF NO: 163 glycoprotein (37%) (5%) (14%) NFSQILPDPSKPSKRS SEQ ID surface  1/22 (5%) 4/22 0/22  4/22 NO: 164 glycoprotein (19%) (0%) (19%) KRSFIEDLLFNKVT SEQ ID surface  5/22 (23%) 8/22 4/22 5/22 NO: 165 glycoprotein (37%) (19%) (23%) EDLLFNKVTLADAGF SEQ ID surface  1/22 (5%) 1/22 1/22  0/22  NO: 166 glycoprotein (5%) (5%) (0%) YGDCLGDIAARDLIC SEQ ID surface  1/22 (5%) 3/22 1/22  0/22  NO: 167 glycoprotein (14%) (5%) (0%) ICAQKFNGLTVLPPLL SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 168 glycoprotein (0%) (0%) (0%) FGAGAALQIPFAMQM SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 169 glycoprotein (0%) (0%) (0%) NGIGVTQNVLYENQK SEQ ID surface  1/22 (5%) 0/22 1/22  1/22  NO: 170 glycoprotein (0%) (5%) (5%) IGKIQDSLSSTASA SEQ ID surface  0/22 (0%) 1/22 1/22  0/22  NO: 171 glycoprotein (5%) (5%) (0%) VLNDILSRLDKVEAE SEQ ID surface  0/22 (0%) 2/22 0/22  0/22  NO: 172 glycoprotein (10%) (0%) (0%) LSRLDKVEAEVQID SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 173 glycoprotein (0%) (0%) (0%) AICHDGKAHFPREGVF SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 174 glycoprotein (0%) (0%) (0%) HWFVTQRNFYEPQIITT SEQ ID surface  0/22 (0%) 0/22 1/22  1/22  NO: 175 glycoprotein (0%) (5%) (5%) VIGIVNNTVYDPLQPEL SEQ ID surface  2/22 (10%) 2/22 0/22  1/22  NO: 176 glycoprotein (10%) (0%) (5%) TVYDPLQPELDSFK SEQ ID surface  1/22 (5%) 1/22 0/22  0/22  NO: 177 glycoprotein (5%) (0%) (0%) PELDSFKEELDKYFKNHT SEQ ID surface  15/22 (69%) 21/22 9/22 18/22 SPDVD NO: 178 glycoprotein (96%) (41%) (82%) SPDVDLGDISGINASVV SEQ ID surface  10/22 (46%) 20/22 6/22 13/22 NO: 179 glycoprotein (91%) (28%) (60%) EIDRLNEVAKNLNESL SEQ ID surface  0/22 (0%) 5/22 0/22  1/22  NO: 180 glycoprotein (23%) (0%) (5%) EVAKNLNESLIDLQ SEQ ID surface  1/22 (5%) 1/22 2/22 1/22  NO: 181 glycoprotein (5%) (10%) (5%) SCGSCCKFDEDDSE SEQ ID surface  0/22 (0%) 0/22 0/22  0/22  NO: 182 glycoprotein (0%) (0%) (0%) FDEDDSEPVLKGVKLH SEQ ID surface  0/22 (0%) 3/22 0/22  1/22  NO: 183 glycoprotein (14%) (0%) (5%) TIGTVTLKQGEIKDAT SEQ ID ORF3a protein 0/22 (0%) 2/22 0/22  1/22  NO: 184 (10%) (0%) (5%) EHDYQIGGYTEKWESGV SEQ ID ORF3a protein 4/22 (19%) 7/22 1/22  2/22 KDCVVL NO: 185 (32%) (5%) (10%) MADSNGTITVEELK SEQ ID membrane  0/22 (0%) 0/22 2/22 2/22 NO: 186 glycoprotein (0%) (10%) (10%) RSMWSFNPETNILLNV SEQ ID membrane  0/22 (0%) 1/22 0/22  0/22  NO: 187 glycoprotein (5%) (0%) (0%) CDIKDLPKEITVATS SEQ ID membrane  0/22 (0%) 8/22 0/22  6/22 NO: 188 glycoprotein (37%) (0%) (28%) KLGASQRVAGDSGFA SEQ ID membrane  0/22 (0%) 1/22 0/22  0/22  NO: 189 glycoprotein (5%) (0%) (0%) QRVAGDSGFAAYSRY SEQ ID membrane  0/22 (0%) 10/22 0/22  4/22 NO: 190 glycoprotein (46%) (0%) (19%) SSGTYEGNSPFHPLA SEQ ID ORF7a protein 1/22 (5%) 1/22 1/22  1/22  NO: 191 (5%) (5%) (5%) LIELCVDEAGSKSPIQYI SEQ ID ORF8 protein 0/22 (0%) 3/22 2/22 1/22  D NO: 192 (14%) (10%) (5%) GSKSPIQYIDIGNYTVSC SEQ ID ORF8 protein 3/22 (14%) 12/22 4/22 3/22 LPF NO: 193 (55%) (19%) (14%) RSKQRRPQGLPNNT SEQ ID nucleocapsid 1/22 (5%) 5/22 0/22  1/22  NO: 194 phosphoprotein (23%) (0%) (5%) DLKFPRGQGVPINT SEQ ID nucleocapsid 0/22 (0%) 0/22 0/22  0/22  NO: 195 phosphoprotein (0%) (0%) (0%) NTNSSPDDQIGYYRR SEQ ID nucleocapsid 0/22 (0%) 0/22 0/22  0/22  NO: 196 phosphoprotein (0%) (0%) (0%) KDHIGTRNPANNAA SEQ ID nucleocapsid 0/22 (0%) 0/22 0/22  0/22  NO: 197 phosphoprotein (0%) (0%) (0%) RGTSPARMAGNGGDA SEQ ID nucleocapsid 1/22 (5%) 3/22 0/22  0/22  NO: 198 phosphoprotein (14%) (0%) (0%) MAGNGGDAALALLLLDR SEQ ID nucleocapsid 0/22 (0%) 2/22 2/22 1/22  NO: 199 phosphoprotein (10%) (10%) (5%) ALLLLDRLNQLESKMSG SEQ ID nucleocapsid 3/22 (14%) 5/22 2/22 5/22 NO: 200 phosphoprotein (23%) (10%) (23%) NQLESKMSGKGQQQQGQ SEQ ID nucleocapsid 12/22 (55%) 18/22 8/22 15/22 TVTKKSA NO: 201 phosphoprotein (82%) (37%) (69%) QTVTKKSAAEASKKPRQK SEQ ID nucleocapsid 2/22 (10%) 7/22 1/22  4/22 NO: 202 phosphoprotein (32%) (5%) (19%) RGPEQTQGNFGDQELI SEQ ID nucleocapsid 6/22 (28%) 3/22 2/22 3/22 NO: 203 phosphoprotein (14%) (10%) (14%) IKLDDKDPNFKDQVIL SEQ ID nucleocapsid 4/22 (19%) 1/22 0/22  0/22  NO: 204 phosphoprotein (5%) (0%) (0%) AYKTFPPTEPKKDKKKK SEQ ID nucleocapsid 2/22 (10%) 5/22 0/22  2/22 NO: 205 phosphoprotein (23%) (0%) (10%) KDKKKKADETQALPQR SEQ ID nucleocapsid 3/22 (14%) 7/22 0/22  5/22 NO: 206 phosphoprotein (32%) (0%) (23%) ADETQALPQRQKKQQTV SEQ ID nucleocapsid 4/22 (19%) 17/22 4/22 9/22 TLLPAADL NO: 207 phosphoprotein (78%) (19%) (41%) TLLPAADLDDFSKQLQQS SEQ ID nucleocapsid 14/22 (64%) 22/22 6/22 18/22 MSSADS NO: 208 phosphoprotein (100%) (28%) (82%) Healthy- Controls_ SARS-CoV- Epitope sequence Asymtomatic Other_CoV China 1 (2003) YRKVLLRKNGNKGA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 46 LLRKNGNKGAGGHSY SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 47 GNKGAGGHSYGADLKSFD SEQ ID 0/12 (0%) 2/12 (17%) 0/10 (0%) 0/10 (0%) L NO: 48 DELGTDPYEDFQEN SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 49 WNTKHSSGVTRELM SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 50 GVYCCREHEHEIAW SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 51 HEIAWYTERSEKSY SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 52 SYELQTPFEIKLAK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 53 AKKFDTFNGECPNF SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 54 CDHCGETSWQTGDF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 55 LTNIFGTVYEKLKPVLD SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 56 GVEFLRDGWEIVKF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 57 TGDLQPLEQPTSEAVEAP SEQ ID 0/12 (0%) 1/12 (9%) 1/10 (10%) 1/10 (10%) LV NO: 58 KGGAPTKVTFGDDTVIEV SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 1/10 (10%) NO: 59 NITFELDERIDKVL SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 60 EFACVVADAVIKTLQPVS SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 61 NKGEDIQLLKSAYENF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 62 YDKLVSSFLEMKSEK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 63 AFYILPSIISNEKQEI SEQ ID 0/12 (0%) 1/12 (9%) 1/10 (10%) 0/10 (0%) NO: 64 LGYVTHGLNLEEAA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 65 THGLNLEEAARYMRS SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 66 TAYNGYLTSSSKTP SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 1/10 (10%) NO: 67 GEVITFDNLKTLLS SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 68 GRYMSALNHTKKWKYPQV SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 69 TVGELGDVRETMSYL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 70 MGTLSYEQFKKGVQIPC SEQ ID 0/12 (0%) 0/12 (0%) 2/10 (20%) 0/10 (0%) NO: 71 FTCASEYTGNYQCGHY SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 72 YQCGHYKHITSKET SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 73 LVPNQPYPNASFDNF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 74 FPDLNGDVVAIDYK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 75 NSFDVLKSEDAQGMDNL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 76 SEEVVENPTIQKDVLECN SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 77 GLAAVNSVPWDTIA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 78 TIANYAKPFLNKVVSTTT SEQ ID 0/12 (0%) 3/12 (25%) 2/10 (20%) 0/10 (0%) NO: 79 FLNKVVSTTTNIVTRC SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 80 VKMFDAYVNTFSST SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 81 FSSTFNVPMEKLKT SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 82 TLVATAEAELAKNVSL SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 83 SAAKKNNLPFKLTCA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 84 VHVMSKHTDFSSEIIG SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 85 DFDTWFSQRGGSYT SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 86 TFDSEYCRHGTCER SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 87 DVLYQPPQTSITSAVL SEQ ID 1/12 (9%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 88 NRFTTTLNDFNLVAMK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 89 LTQDHVDILGPLSA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 90 GMNGRTILGSALLED SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 91 QGLLPPKNSIDAFKLNIK SEQ ID 0/12 (0%) 2/12 (17%) 0/10 (0%) 0/10 (0%) NO: 92 KLKKSLNVAKSEFDRD SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 93 LRANSAVKLQNNELS SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 94 AVKLQNNELSPVALR SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 95 CAAGTTQTACTDDN SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 96 LALLSDLQDLKWARF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 97 ATVRLQAGNATEVPA SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 98 VKMLCTHTGTGQAITV SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 99 KYVQIPTTCANDPV SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 100 RVCGVSAARLTPCGT SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 101 AVAKHDFFKFRIDG SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 102 GDFIQTTPGSGVPV SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 103 QTYHPNCVNCLDDR SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 104 ALTNNVAFQTVKPGNF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 105 GFFKEGSSVELKHF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 106 QLLFVVEVVDKYFDCYDG SEQ ID 0/12 (0%) 3/12 (25%) 1/10 (10%) 0/10 (0%) GC NO: 107 SLYVKPGGTSSGDATTAY SEQ ID 0/12 (0%) 0/12 (0%) 2/10 (20%) 0/10 (0%) A NO: 108 NRDVDTDFVNEFYAYL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 109 SQGLVASIKNFKSV SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 110 QHTMLVKQGDDYVY SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 111 VDDIVKTDGTLMIE SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 112 AIDAYPLTKHPNQE SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 113 GCDVTDVTQLYLGG SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 114 GLYKNTCVGSDNVT SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 115 NTCVGSDNVTDFNAIATC SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) DW NO: 116 YQKVGMQKYSTLQG SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 117 STLQGPPGTGKSHF SEQ ID 1/12 (9%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 118 ARARVECFDKFKVNS SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 119 DPAQLPAPRTLLTK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 120 PRTLLTKGTLEPEY SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 121 IFTQTTETAHSCNV SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 122 GCHATREAVGTNLP SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 123 VQMLSDTLKNLSDR SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 124 FDYVYNPFMIDVQQWG SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 125 MIDVQQWGFTGNLQ SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 126 TGNLQSNHDLYCQVH SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 127 LADKFPVLHDIGNPKAIK SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 1/10 (10%) CVP NO: 128 FYSYATHSDKFTDGV SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 129 FDTRVLSNLNLPGCD SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 130 QVVSDIDYVPLKSATC SEQ ID 1/12 (9%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 131 PSVGPKQASLNGVTL SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 132 GVVQQLPETYFTQSRN SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 133 AMDEFIERYKLEGY SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 134 LAKRFKESPFELED SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 1/10 (10%) NO: 135 FITDAQTGSSKCVCSVID SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 136 IKSQDLSVVSKVVKV SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 137 YPKLQSSQAWQPGV SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 138 QRMLLEKCDLQNYG SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 139 HFGAGSDKGVAPGTA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 140 ITEHSWNADLYKLM SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 141 LSSYSLFDMSKFPLKL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 142 SLLLCRMNSRNYIA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 143 QCVNLTTRTQLPPA SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 144 TQLPPAYTNSFTRGVYYP SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) D NO: 145 FHAIHVSGTNGTKRF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 146 NLVRDLPQGFSALEPLV SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 147 LALHRSYLTPGDSSSGWT SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 148 KYNENGTITDAVDCALDP SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) LS NO: 149 IYQAGSTPCNGVEGFNCY SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 150 VLSFELLHAPATVC SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 151 LLHAPATVCGPKKST SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 152 VKNKCVNFNFNGLT SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 153 TESNKKFLPFQQFGRDIA SEQ ID 3/12 (25%) 0/12 (0%) 0/10 (0%) 0/10 (0%) D NO: 154 TDAVRDPQTLEILDITPC SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) S NO: 155 QVAVLYQDVNCTEVPVAI SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) HADQLTPTW NO: 156 IHADQLTPTWRVYSTGSN SEQ ID 1/12 (9%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 157 YSTGSNVFQTRAGCL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 158 CASYQTQTNSPRRARSV SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 159 ARSVASQSIIAYTMSLGA SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) ENSVA NO: 160 PVSMTKTSVDCTMY SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 161 RALTGIAVEQDKNTQEVF SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) AQV NO: 162 FAQVKQIYKTPPIKDFGG SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) FNF NO: 163 NFSQILPDPSKPSKRS SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 164 KRSFIEDLLFNKVT SEQ ID 1/12 (9%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 165 EDLLFNKVTLADAGF SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 166 YGDCLGDIAARDLIC SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 167 ICAQKFNGLTVLPPLL SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 168 FGAGAALQIPFAMQM SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 169 NGIGVTQNVLYENQK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 170 IGKIQDSLSSTASA SEQ ID 0/12 (0%) 1/12 (9%) 1/10 (10%) 0/10 (0%) NO: 171 VLNDILSRLDKVEAE SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 172 LSRLDKVEAEVQID SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 173 AICHDGKAHFPREGVF SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 174 HWFVTQRNFYEPQIITT SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 175 VIGIVNNTVYDPLQPEL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 176 TVYDPLQPELDSFK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 177 PELDSFKEELDKYFKNHT SEQ ID 8/12 (67%) 1/12 (9%) 0/10 (0%) 1/10 (10%) SPDVD NO: 178 SPDVDLGDISGINASVV SEQ ID 0/12 (0%) 1/12 (9%) 2/10 (20%) 1/10 (10%) NO: 179 EIDRLNEVAKNLNESL SEQ ID 1/12 (9%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 180 EVAKNLNESLIDLQ SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 181 SCGSCCKFDEDDSE SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) NO: 182 FDEDDSEPVLKGVKLH SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 183 TIGTVTLKQGEIKDAT SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 184 EHDYQIGGYTEKWESGVK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) DCVVL NO: 185 MADSNGTITVEELK SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 186 RSMWSFNPETNILLNV SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 187 CDIKDLPKEITVATS SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 188 KLGASQRVAGDSGFA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 189 QRVAGDSGFAAYSRY SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 190 SSGTYEGNSPFHPLA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 191 LIELCVDEAGSKSPIQYI SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) D NO: 192 GSKSPIQYIDIGNYTVSC SEQ ID 0/12 (0%) 1/12 (9%) 0/10 (0%) 0/10 (0%) LPF NO: 193 RSKQRRPQGLPNNT SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 194 DLKFPRGQGVPINT SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 195 NTNSSPDDQIGYYRR SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 1/10 (10%) NO: 196 KDHIGTRNPANNAA SEQ ID 0/12 (0%) 0/12 (0%) 1/10 (10%) 0/10 (0%) NO: 197 RGTSPARMAGNGGDA SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 198 MAGNGGDAALALLLLDR SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 199 ALLLLDRLNQLESKMSG SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 200 NQLESKMSGKGQQQQGQT SEQ ID 7/12 (59%) 0/12 (0%) 0/10 (0%) 0/10 (0%) VTKKSA NO: 201 QTVTKKSAAEASKKPRQK SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 202 RGPEQTQGNFGDQELI SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 203 IKLDDKDPNFKDQVIL SEQ ID 0/12 (0%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 204 AYKTFPPTEPKKDKKKK SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 205 KDKKKKADETQALPQR SEQ ID 3/12 (25%) 0/12 (0%) 0/10 (0%) 0/10 (0%) NO: 206 ADETQALPQRQKKQQTVT SEQ ID 2/12 (17%) 0/12 (0%) 0/10 (0%) 0/10 (0%) LLPAADL NO: 207 TLLPAADLDDFSKQLQQS SEQ ID 7/12 (59%) 1/12 (9%) 0/10 (0%) 0/10 (0%) MSSADS NO: 208

TABLE 5 IgG linear IgG epitopes for detection of HKU-1, NL63, OC43, 229E, SARS-CoV-1.  Sequences (aa) are based on proteome of reference sequences from NCBI Controls   Other Epitope Protein SARS- Human Virus name sequences name CoV-1 CoV Healthy  1. Human  SEQ ID  MVNPSKGSSGLLIT Nucleo- 0/10  4/12  0/10 (0%) coronavirus NO: 209 KLT capsid (0%) (34%) HKU1 phospho- protein  2. Human  SEQ ID  VKQLPEQFDSLNLS Nucleo- 0/10  2/12  1/10 (10%) coronavirus NO: 210 AGTQ capsid (0%) (17%) HKU1 phospho- protein  3. Human  SEQ ID  SLLATLDDPYVEDS Nucleo- 1/10  2/12  0/10 (0%) coronavirus NO: 211 VA capsid (10%) (17%) HKU1 phospho- protein  4. Human  SEQ ID  MNKSFLPQFTSDQA Membrane 5/10  4/12  6/10 (60%) coronavirus NO: 212 V glyco- (50%) (34%) HKU1 protein  5. Human  SEQ ID  WLNNFNVPSPLNW Spike  0/10  2/12  0/10 (0%) coronavirus NO: 213 ERKI glyco- (0%) (17%) HKU1 protein  6. Human  SEQ ID  ISASYRFVTFEPFNV Spike  2/10  4/12  2/10 (20%) coronavirus NO: 214 S glyco- (20%) (34%) HKU1 protein  7. Human  SEQ ID  SRSFFEDLLFDKVK Spike  0/10  1/12  1/10 (10%) coronavirus NO: 215 LS glyco- (0%) (9%) HKU1 protein  8. Human  SEQ ID  VDKQVILVSNNKE orf1ab  0/10  1/12  1/10 (10%) coronavirus NO: 216 DFDIIQ poly- (0%) (9%) HKU1 protein  9. Human  SEQ ID  LLHSKVKVVPNLY orf1ab  1/10  3/12  2/10 (20%) coronavirus NO: 217 VVV poly- (10%) (25%) HKU1 protein 10. Human  SEQ ID  FDLVQYDFTDYKL orf1ab  1/10  1/12  0/10 (0%) coronavirus NO: 218 EL poly- (10%) (9%) HKU1 protein 11. Human  SEQ ID  PASQVIVNNYDKSA orf1ab  0/10  2/12  1/10 (10%) coronavirus NO: 219 GY poly- (0%) (17%) HKU1 protein 12. Human  SEQ ID  CTGSPYIDDFNKIAS orf1ab  0/10  1/12  0/10 (0%) coronavirus NO: 220 poly- (0%) (9%) HKU1 protein 13. Human  SEQ ID  CWETGKVKPPLNK orf1ab  1/10  1/12  2/10 (20%) coronavirus NO: 221 NYVFTG poly- (10%) (9%) HKU1 protein 14. Human  SEQ ID  AKVRVDCYDKFKI orf1ab  1/10  2/12  0/10 (0%) coronavirus NO: 222 NDTTCKY poly- (10%) (17%) HKU1 protein 15. Human  SEQ ID  ISPYNSQNYVAKRV orf1ab  0/10  1/12  1/10 (10%) coronavirus NO: 223 LGVQ poly- (0%) (9%) HKU1 protein 16. Human  SEQ ID  EFRDYFNNNTDSIV orf1ab  1/10  2/12  2/10 (20%) coronavirus NO: 224 poly- (10%) (17%) NL63 protein 17. Human  SEQ ID  RKASGLFDAIWDAF orf1ab  0/10  1/12  2/10 (20%) coronavirus NO: 225 VAAI poly- (0%) (9%) NL63 protein 18. Human  SEQ ID  NVGEPIISQPSKLLN orf1ab  2/10  1/12  0/10 (0%) coronavirus NO: 226 G poly- (20%) (9%) NL63 protein 19. Human  SEQ ID  ISYLNRARGSSAAR orf1ab  1/10  2/12  0/10 (0%) coronavirus NO: 227 LEPC poly- (10%) (17%) NL63 protein 20. Human  SEQ ID  CIKACEVVVTNLNK orf1ab  1/10  3/12  1/10 (10%) coronavirus NO: 228 SAGWPLNKFGKA poly- (10%) (25%) NL63 protein 21. Human  SEQ ID  VFINKYGLEDFNFE orf1ab  0/10  1/12  0/10 (0%) coronavirus NO: 229 H poly- (0%) (9%) NL63 protein 22. Human  SEQ ID  TVLNASIPESKPLA Nucleo- 7/10  3/12  6/10 (60%) coronavirus NO: 230 DDDSA capsid (70%) (25%) NL63 phospho- protein 23. Human  SEQ ID  ASQEGVLSEREKLL Membrane 0/10  2/12  0/10 (0%) coronavirus NO: 231 HLI glyco- (0%) (17%) NL63 protein 24. Human  SEQ ID  QFGLQDGFYSANFL Spike  3/10  4/12  6/10 (60%) coronavirus NO: 232 DDNVL glyco- (30%) (34%) NL63 protein 25. Human  SEQ ID  CDCGSTKLPYYEFE Spike  0/10  3/12  7/10 (70%) coronavirus NO: 233 KVHVQ glyco- (0%) (25%) NL63 protein 26. Human  SEQ ID  FDLVQYDFTDYKL nsp9 1/10  1/12  0/10 (0%) coronavirus NO: 234 EL (10%) (9%) OC43 27. Human  SEQ ID  SQVIVNNYDKSAG nsp9 0/10  2/12  0/10 (0%) coronavirus NO: 235 YPF (0%) (17%) OC43 28. Human  SEQ ID  LSTCDGQKFTDESF nsp9 0/10  2/12  1/10 (10%) coronavirus NO: 236 YKNMY (0%) (17%) OC43 29. Human  SEQ ID  FPLKLAGTAVINLR nsp13 0/10  2/12  0/10 (0%) coronavirus NO: 237 AD (0%) (17%) OC43 30. Human  SEQ ID  HATRDSIGTNFPLQ nsp11 0/10  1/12  1/10 (10%) coronavirus NO: 238 L (0%) (9%) OC43 31. Human  SEQ ID  CTGSPYIDDFNRIAS nsp10 0/10  1/12  0/10 (0%) coronavirus NO: 239 (0%) (9%) OC43 32. Human  SEQ ID  AKVRVECYDKFKI nsp10 1/10  3/12  0/10 (0%) coronavirus NO: 240 NDTTRKYV (10%) (25%) OC43 33. Human  SEQ ID  ASSRSAIEDLLFDK Spike  5/10  3/12  5/10 (50%) coronavirus NO: 241 VKLS glyco- (50%) (25%) OC43 protein 34. Human  SEQ ID  SQVIVNNYDKSAG orf1ab  0/10  2/12  0/10 (0%) coronavirus NO: 242 YPF poly- (0%) (17%) OC43 protein 35. Human  SEQ ID  LSTCDGQKFTDESF orf1ab  0/10  2/12  1/10 (10%) coronavirus NO: 243 YKNMY poly- (0%) (17%) OC43 protein 36. Human  SEQ ID  AETQKATEEAFKQS orf1ab  0/10  2/12  0/10 (0%) coronavirus NO: 244 YA poly- (0%) (17%) OC43 protein 37. Human  SEQ ID  SWEIGKVKPPLNKN orf1ab  1/10  1/12  3/10 (30%) coronavirus NO: 245 YVFTG poly- (10%) (9%) OC43 protein 38. Human  SEQ ID  AKVRVECYDKFKI orf1ab  2/10  3/12  1/10 (10%) coronavirus NO: 246 NDTTRKYV poly- (20%) (25%) OC43 protein 39. Human  SEQ ID  HATRDSIGTNFPLQ orf1ab  0/10  2/12  1/10 (10%) coronavirus NO: 247 L poly- (0%) (17%) OC43 protein 40. Human  SEQ ID  DVIDNEIIVKPNISL orf1ab  3/10  4/12  3/10 (30%) coronavirus NO: 248 CVPL poly- (30%) (34%) 229E protein 41. Human  SEQ ID  KHSDFGLGDLVDS orf1ab  0/10  1/12  2/10 (20%) coronavirus NO: 249 YFEND poly- (0%) (9%) 229E protein 42. Human  SEQ ID  CITSREVVVTNLNK orf1ab  0/10  3/12  0/10 (0%) coronavirus NO: 250 SA poly- (0%) (25%) 229E protein 43. Human  SEQ ID  DVIDNEIIVKPNISL orf1ab  3/10  4/12  3/10 (30%) coronavirus NO: 251 CVPL poly- (30%) (34%) 229E protein 44. Human  SEQ ID  KHSDFGLGDLVDS orf1ab  0/10  1/12  2/10 (20%) coronavirus NO: 252 YFEND poly- (0%) (9%) 229E protein 45. SARS-CoV-1 SEQ ID  LYEQVVMDYLDNL orf1ab 7/10  3/12  4/10 (40%) NO: 253 KPRVEAPKQEEPPN poly- (70%) (25%) TED protein 46. SARS-CoV-1 SEQ ID  VSVSSPDAVTTYNG nsp3 3/10  0/12  2/10 (20%) NO: 254 YLTSSSKTSEEHF (30%) (0%) 47. SARS-CoV-1 SEQ ID  LGDELGTDPIEDYE orf1ab  3/10  2/12  3/10 (30%) NO: 255 QNWNTKHGSGALR poly- (30%) (17%) protein 48. SARS-CoV-1 SEQ ID  FTLRSITAQPVKIDN Orf3 4/10  0/12  0/10 (0%) NO: 256 ASPASTVHATA (40%) (0%) 49. SARS-CoV-1 SEQ ID  TIVVTEGDGISTPKL Orf3 5/10  1/12  0/10 (0%) NO: 257 KEDYQIGGYSEDR (50%) (9%) 50. SARS-CoV-1 SEQ ID  SSGVANPAMDPIYD Orf3 4/10  1/12  1/10 (10%) NO: 258 EPTTTTSVPL (40%) (9%) 51. SARS-CoV-1 SEQ ID  MADNGTITVEELK membrane 6/10  1/12  0/10 (0%) NO: 259 QLLEQWNLVIG glyco- (60%) (9%) protein 52. SARS-CoV-1 SEQ ID  TPKDHIGTRNPNNN Nucleo- 5/10  2/12  0/10 (0%) NO: 260 AATVLQLPQGTTLP capsid (50%) (17%) KGFYAEGSRGGS phospho- protein 53. SARS-CoV-1 SEQ ID  FSLDVSEKSGNFKH spike  4/10  0/12  1/10 (10%) NO: 261 LREFVF glyco- (40%) (0%) protein Asymp- Mild Severe to- Mild di- Severe di- Asympto- matic di- sease di- sease Epitope matic (Sec- sease (sec- sease (sec- sequences Protein name (first) ond) (first) ond) (first) ond)  1. MVNPSKGSSGLLIT SEQ ID  Nucleocapsid 0/6 (0%) 0/6  0/22 1/22 0/22 0/22 KLT NO: 209 phosphoprotein (0%) (0%) (5%) (0%) (0%)  2. VKQLPEQFDSLNLS SEQ ID  Nucleocapsid 2/6 (34%) 0/6  6/22 6/22 10/22 11/22 AGTQ NO: 210 phosphoprotein (0%) (28%) (28%) (46%) (50%)  3. SLLATLDDPYVEDS SEQ ID  Nucleocapsid 0/6 (0%) 0/6  4/22 1/22 5/22 10/22 VA NO: 211 phosphoprotein (0%) (19%) (5%) (23%) (46%)  4. MNKSFLPQFTSDQA SEQ ID  Membrane 6/6  4/6  19/22 15/22 18/22 21/22 V NO: 212 glycoprotein (100%) (67%) (87%) (69%) (82%) (96%)  5. WLNNFNVPSPLNWE SEQ ID  Spike 0/6 (0%) 0/6  0/22 0/22 1/22 0/22 RKI NO: 213 glycoprotein (0%) (0%) (0%) (5%) (0%)  6. ISASYRFVTFEPFNV SEQ ID  Spike 2/6 (34%) 0/6  11/22 10/22 11/22 10/22 S NO: 214 glycoprotein (0%) (50%) (46%) (50%) (46%)  7. SRSFFEDLLFDKVKL SEQ ID  Spike 0/6 (0%) 1/6  5/22 3/22 3/22 4/22 S NO: 215 glycoprotein (17%) (23%) (14%) (14%) (19%)  8. VDKQVILVSNNKED SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 2/22 7/22 11/22 FDIIQ NO: 216 polyprotein (0%) (0%) (10%) (32%) (50%)  9. LLHSKVKVVPNLYV SEQ ID  orf1ab 0/6 (0%) 0/6  1/22 0/22 2/22 2/22 VV NO: 217 polyprotein (0%) (5%) (0%) (10%) (10%) 10. FDLVQYDFTDYKLE SEQ ID  orf1ab 0/6 (0%) 0/6  4/22 2/22 7/22 11/22 L NO: 218 polyprotein (0%) (19%) (10%) (32%) (50%) 11. PASQVIVNNYDKSA SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 0/22 4/22 3/22 GY NO: 219 polyprotein (0%) (0%) (0%) (19%) (14%) 12. CTGSPYIDDFNKIAS SEQ ID  orf1ab 0/6 (0%) 1/6  6/22 10/22 11/22 14/22 NO: 220 polyprotein (17%) (28%) (46%) (50%) (64%) 13. CWETGKVKPPLNK SEQ ID  orf1ab 1/6 (17%) 2/6  4/22 5/22 4/22 5/22 NYVFTG NO: 221 polyprotein (34%) (19%) (23%) (19%) (23%) 14. AKVRVDCYDKFKIN SEQ ID  orf1ab 2/6 (34%) 2/6  2/22 1/22 4/22 4/22 DTTCKY NO: 222 polyprotein (34%) (10%) (5%) (19%) (19%) 15. ISPYNSQNYVAKRV SEQ ID  orf1ab 0/6 (0%) 0/6  2/22 2/22 5/22 6/22 LGVQ NO: 223 polyprotein (0%) (10%) (10%) (23%) (28%) 16. EFRDYFNNNTDSIV SEQ ID  orf1ab 0/6 (0%) 0/6  3/22 3/22 6/22 8/22 NO: 224 polyprotein (0%) (14%) (14%) (28%) (37%) 17. RKASGLFDAIWDAF SEQ ID  orf1ab 0/6 (0%) 0/6  2/22 1/22 0/22 1/22 VAAI NO: 225 polyprotein (0%) (10%) (5%) (0%) (5%) 18. NVGEPIISQPSKLLN SEQ ID  orf1ab 1/6 (17%) 1/6  0/22 0/22 2/22 1/22 G NO: 226 polyprotein (17%) (0%) (0%) (10%) (5%) 19. ISYLNRARGSSAARL SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 0/22 0/22 0/22 EPC NO: 227 polyprotein (0%) (0%) (0%) (0%) (0%) 20. CIKACEVVVTNLNK SEQ ID  orf1ab 0/6 (0%) 0/6  1/22 2/22 1/22 3/22 SAGWPLNKFGKA NO: 228 polyprotein (0%) (5%) (10%) (5%) (14%) 21. VFINKYGLEDFNFE SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 0/22 5/22 5/22 H NO: 229 polyprotein (0%) (0%) (0%) (23%) (23%) 22. TVLNASIPESKPLAD SEQ ID  Nucleocapsid 3/6 (50%) 3/6  15/22 17/22 15/22 21/22 DDSA NO: 230 phosphoprotein (50%) (69%) (78%) (69%) (96%) 23. ASQEGVLSEREKLL SEQ ID  Membrane 0/6 (0%) 0/6  0/22 1/22 1/22 2/22 HLI NO: 231 glycoprotein (0%) (0%) (5%) (5%) (10%) 24. QFGLQDGFYSANFL SEQ ID  Spike 1/6 (17%) 1/6  6/22 7/22 8/22 13/22 DDNVL NO: 232 glycoprotein (17%) (28%) (32%) (37%) (60%) 25. CDCGSTKLPYYEFE SEQ ID  Spike 1/6 (17%) 2/6  8/22 6/22 12/22 15/22 KVHVQ NO: 233 glycoprotein (34%) (37%) (28%) (55%) (69%) 26. FDLVQYDFTDYKLE SEQ ID  nsp9 0/6 (0%) 0/6  4/22 2/22 7/22 12/22 L NO: 234 (0%) (19%) (10%) (32%) (55%) 27. SQVIVNNYDKSAGY SEQ ID  nsp9 0/6 (0%) 0/6  0/22 0/22 6/22 9/22 PF NO: 235 (0%) (0%) (0%) (28%) (41%) 28. LSTCDGQKFTDESF SEQ ID  nsp9 0/6 (0%) 0/6  0/22 1/22 1/22 6/22 YKNMY NO: 236 (0%) (0%) (5%) (5%) (28%) 29. FPLKLAGTAVINLR SEQ ID  nsp13 0/6 (0%) 0/6  0/22 0/22 0/22 0/22 AD NO: 237 (0%) (0%) (0%) (0%) (0%) 30. HATRDSIGTNFPLQL SEQ ID  nsp11 0/6 (0%) 0/6  5/22 6/22 2/22 6/22 NO: 238 (0%) (23%) (28%) (10%) (28%) 31. CTGSPYIDDFNRIAS SEQ ID  nsp10 0/6 (0%) 0/6  2/22 2/22 3/22 5/22 NO: 239 (0%) (10%) (10%) (14%) (23%) 32. AKVRVECYDKFKIN SEQ ID  nsp10 1/6 (17%) 2/6  1/22 3/22 2/22 5/22 DTTRKYV NO: 240 (34%) (5%) (14%) (10%) (23%) 33. ASSRSAIEDLLFDKV SEQ ID  Spike 1/6 (17%) 3/6  13/22 14/22 13/22 16/22 KLS NO: 241 glycoprotein (50%) (60%) (64%) (60%) (73%) 34. SQVIVNNYDKSAGY SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 0/22 0/22 0/22 PF NO: 242 polyprotein (0%) (0%) (0%) (0%) (0%) 35. LSTCDGQKFTDESF SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 1/22 1/22 6/22 YKNMY NO: 243 polyprotein (0%) (0%) (5%) (5%) (28%) 36. AETQKATEEAFKQS SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 0/22 0/22 0/22 YA NO: 244 polyprotein (0%) (0%) (0%) (0%) (0%) 37. SWEIGKVKPPLNKN SEQ ID  orf1ab 2/6 (34%) 2/6  5/22 4/22 5/22 6/22 YVFTG NO: 245 polyprotein (34%) (23%) (19%) (23%) (28%) 38. AKVRVECYDKFKIN SEQ ID  orf1ab 1/6 (17%) 2/6  1/22 3/22 5/22 8/22 DTTRKYV NO: 246 polyprotein (34%) (5%) (14%) (23%) (37%) 39. HATRDSIGTNFPLQL SEQ ID  orf1ab 0/6 (0%) 0/6  6/22 7/22 2/22 6/22 NO: 247 polyprotein (0%) (28%) (32%) (10%) (28%) 40. DVIDNEIIVKPNISLC SEQ ID  orf1ab 1/6 (17%) 0/6  9/22 11/22 16/22 19/22 VPL NO: 248 polyprotein (0%) (41%) (50%) (73%) (87%) 41. KHSDFGLGDLVDSY SEQ ID  orf1ab 0/6 (0%) 1/6  10/22 7/22 15/22 19/22 FEND NO: 249 polyprotein (17%) (46%) (32%) (69%) (87%) 42. CITSREVVVTNLNKS SEQ ID  orf1ab 0/6 (0%) 0/6  0/22 0/22 2/22 1/22 A NO: 250 polyprotein (0%) (0%) (0%) (10%) (5%) 43. DVIDNEIIVKPNISLC SEQ ID  orf1ab 1/6 (17%) 0/6  10/22 11/22 16/22 19/22 VPL NO: 251 polyprotein (0%) (46%) (50%) (73%) (87%) 44. KHSDFGLGDLVDSY SEQ ID  orf1ab 0/6 (0%) 0/6  12/22 7/22 15/22 19/22 FEND NO: 252 polyprotein (0%) (55%) (32%) (69%) (87%) 45. LYEQVVMDYLDNL SEQ ID  orf1ab 3/6 (50%) 4/6  12/22 14/22 13/22 14/22 KPRVEAPKQEEPPN NO: 253 polyprotein (67%) (55%) (64%) (60%) (64%) TED 46. VSVSSPDAVTTYNG SEQ ID  nsp3 0/6 (0%) 0/6  10/22 10/22 7/22 12/22 YLTSSSKTSEEHF NO: 254 (0%) (46%) (46%) (32%) (55%) 47. LGDELGTDPIEDYE SEQ ID  orf1ab 0/6 (0%) 0/6  10/22 13/22 17/22 18/22 QNWNTKHGSGALR NO: 255 polyprotein (0%) (46%) (60%) (78%) (82%) 48. FTLRSITAQPVKIDN SEQ ID  Orf3 1/6 (17%) 1/6  0/22 1/22 0/22 1/22 ASPASTVHATA NO: 256 (17%) (0%) (5%) (0%) (5%) 49. TIVVTEGDGISTPKL SEQ ID  Orf3 0/6 (0%) 0/6  0/22 0/22 2/22 1/22 KEDYQIGGYSEDR NO: 257 (0%) (0%) (0%) (10%) (5%) 50. SSGVANPAMDPIYD SEQ ID  0rf3 1/6 (17%) 0/6  0/22 2/22 4/22 8/22 EPTTTTSVPL NO: 258 (0%) (0%) (10%) (19%) (37%) 51. MADNGTITVEELKQ SEQ ID  membrane 0/6 (0%) 1/6  1/22 15/22 4/22 13/22 LLEQWNLVIG NO: 259 glycoprotein (17%) (5%) (69%) (19%) (60%) 52. TPKDHIGTRNPNNN SEQ ID  Nucleocapsid 2/6 (34%) 3/6  10/22 16/22 15/22 22/22 AATVLQLPQGTTLP NO: 260 phosphoprotein (50%) (46%) (73%) (69%) (100%) KGFYAEGSRGGS 53. FSLDVSEKSGNFKH SEQ ID  spike 0/6 (0%) 0/6  1/22 2/22 1/22 4/22 LREFVF NO: 261 glycoprotein (0%) (5%) (10%) (5%) (19%)

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1. An isolated peptide that is strongly reactive with, and specific for antibodies to SARS-CoV-2, wherein the peptide comprises the amino acid sequence chosen from the group consisting of SEQ ID NOs: 1-45.
 2. (canceled)
 3. (canceled)
 4. The collection of isolated peptides of claim 7, wherein the peptides range in length from about 6 amino acids to 50 amino acids.
 5. The collection of isolated peptides of claim 7, wherein one or more of the isolated peptides is conjugated to a ligand, avidin, streptavidin, neutravidin, serum albumin, keyhole limpet hemocyanin (KLH), an enzyme, a metallic nanoparticle or nanoshell or is biotinylated.
 6. The collection of isolated peptides of claim 7, wherein one or more of the isolated peptides is immobilized to a solid support.
 7. A collection of isolated peptides that are strongly reactive with, and specific for antibodies to SARS-CoV-2, comprising: a peptide or peptide chosen from the group consisting of SEQ ID NOs: 1-45; and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of SEQ ID NOs: 1-45; or combinations thereof.
 8. A peptide microarray comprising the collection of isolated peptides of claim
 7. 9. A method for the differential serological detection of serological detection of exposure to and/or infection by SARS-CoV-2 in a sample, comprising: a. contacting the sample with the collection of peptides of claim 7, under conditions sufficient to allow binding of antibody(ies) to the peptide(s); and b. detecting formation of an antibody-peptide complex comprising said one or more peptides in the collection, wherein formation of the complex is indicative of an antibody to an epitope of a SARS-CoV-2 antigen being present in the sample.
 10. The method of claim 9, wherein the sample is from a subject.
 11. The method of claim 10, wherein the subject is a test subject who has been administered a corona virus vaccine or immunomodulatory agent.
 12. The method of claim 9, wherein the sample is chosen from the group consisting of nasopharyngeal aspirate, blood, cerebrospinal fluid, saliva, serum, plasma, urine, sputum, bronchial lavage, pericardial fluid, and peritoneal fluid.
 13. The method of claim 9, wherein the sample is chosen from the group consisting of cells, cell culture, cell culture medium and compositions used for the development of pharmaceutical and therapeutic agents.
 14. The method of claim 9, wherein the collection of peptides is immobilized to a solid support.
 15. The method of claim 9, further comprising contacting the sample with a collection of peptides comprising a peptide or peptides chosen from the group consisting of SEQ ID NOs: 209-261; and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of SEQ ID NOs: 209-261; or combinations thereof, and further detecting the formation of an antibody-peptide complex comprising said one or more peptides in the collection, wherein formation of the complex is indicative of an antibody to an epitope of another human coronavirus other than SARS-CoV-2 being present in the sample
 16. A method for the differential serological detection of exposure to and/or infection by SARS-CoV-2 in a sample, comprising: a. contacting the sample with at least one peptide comprising the amino acid sequence chosen from the group consisting of SEQ ID NOs: 1-45, under conditions sufficient to allow binding of antibody(ies) in the sample to the peptide(s); and b. detecting formation of an antibody-peptide complex comprising said one or more peptides, wherein formation of the complex is indicative of an antibody to an epitope of SARS-CoV-2 being present in the sample.
 17. The method of claim 16, wherein the sample is from a subject.
 18. The method of claim 17, wherein the subject is a test subject which has been administered a SARS-CoV-2 vaccine or immunomodulatory agent.
 19. The method of claim 16, wherein the sample is chosen from the group consisting of nasopharyngeal aspirate, blood, cerebrospinal fluid, saliva, serum, plasma, urine, sputum, bronchial lavage, pericardial fluid, and peritoneal fluid.
 20. (canceled)
 21. The method of claim 16, further comprising contacting the sample with at least one peptide comprising the amino acid sequence chosen from the group consisting of SEQ ID NOs: 209-261 and further detecting the formation of an antibody-peptide complex comprising said peptide(s), wherein formation of the complex is indicative of an antibody to an epitope of another human coronavirus other than SARS-CoV-2 being present in the sample
 22. (canceled)
 23. A kit comprising the collection of isolated peptides of claim
 7. 24. (canceled)
 25. The kit of claim 23, further comprising a collection of peptides comprising a peptide or peptides chosen from the group consisting of SEQ ID NOs: 209-261; and/or a collection or set of peptides shifted one residue across at least one peptide chosen from the group consisting of SEQ ID NOs: 209-261; or combinations thereof. 